WO2013074833A1 - Capture probe and assay for analysis of fragmented nucleic acids - Google Patents

Abstract

Disclosed is an efficient and scalable method for targeted resequencing and variant identification of nucleic acids such as genomic DNA found in single stranded, fragmented form, such as in a clinical sample of formalin- fixed, paraffin-embedded (FFPE) tissue. The method uses a large number of capture probes mixed with the sample in the presence of a 5' to 3' exonuc lease, a 3' to 5' exonuclease, a ligase, and a universal amplification oligonucleotide that hybridizes to the various capture probes. The nucleases act on ssDNA, not dsDNA. A single stranded circle is formed by the ligase, and is then amplified to produce a population (library) of double stranded linear DNA molecules that are suitable for sequencing. It is shown that the library produces a high degree of fidelity to the original sample, and predictable base changes are shown.

Description

CAPTURE PROBE AND ASSAY FOR ANALYSIS OF FRAGMENTED NUCLEIC

ACIDS

Inventors: Hua Xu, Georges Natsoulis, and Hanlee Ji

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Patent Application

No. 61/560,412 filed on November 16, 2011, which is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under contracts 2P01HG000205 and R21CA 140089-01 Al awarded by the National Institutes of Health. The Government has certain rights in this invention.

REFERENCE TO SEQUENCE LISTING, COMPUTER PROGRAM,

OR COMPACT DISK

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 26, 2012, is named 381596PCT.txt and is 828,326 bytes in size.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates to the field of nucleic analysis, and, more particularly, to methods for contacting fragmented nucleic acids, such as genomic DNA with probes and enzymes whereby selected portions of the genomic DNA are amplified and assayed.

RELATED ART

Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. That is, individual parts or methods used in the present invention may be described in greater detail in the materials discussed below, which materials may provide further guidance to those skilled in the art for making or using certain aspects of the present invention as claimed. The discussion below should not be construed as an admission as to the relevance of the information to any claims herein or the prior art effect of the material described. Next generation DNA sequencing (NGS) has revolutionized genetics by enabling one to routinely sequence human genomes, either in their entirety or specific subsets. While NGS advances have dramatically increased our ability to identify disease-related genetic variants, the widespread application of NGS-based approaches to clinical populations faces some limitations. Citing an example, NGS-based discovery of cancer mutations for large translational and clinical studies is severely restricted by the availability of clinical samples from which one can extract high quality genomic DNA. The vast majority of cancers samples like gastric and colorectal cancer are processed with formalin fixed paraffin embedding (FFPE) of tissues. For clinical pathology laboratories, this is a preservation method because (1) it maintains morphological features of the tumor, (2) enables

histopathologic examination with a number of staining processes and (3) can be stored indefinitely at room temperature. However, the fixation process causes irreversible damage to the sample genomic DNA via cross linkages and increased fragmentation. As a result, genomic DNA extracted from FFPE material is often of poor quality. Furthermore, FFPE- extracted genomic DNA is generally in a single stranded form because of the need for high temperature incubations to melt the paraffin. Therefore, the analysis of FFPE-derived genomic DNA using PCR-based assays is difficult. Overall, these issues restrict our ability to conduct clinical population genetic studies and genetic diagnostic development using these valuable samples. A variety of methods have been developed to enrich specific regions of the human genome. These include in-solution hybridization enrichment, multiplexed-PCR and targeted circularization approaches. Hybrid selection methods apply immobilized oligonucleotides on either microarrays [1-3] or beads [4] to enrich genomic targets from a modified DNA sample. In multiplex-PCR [5], complex primer sets can be utilized to selectively amplify targeted regions prior to modifying DNA for the sequencer. Highly parallel simplex PCR reactions can be conducted with microdroplet technology [6]. In-solution oligonucleotide-based approaches such as molecular inversion probes (MIPs) capture targets by DNA synthesis across the target and ligation that result in circularization of the capture oligonucleotides [7, 8]. Citing another in-solution approach, targeted genomic circularization (TGC) directly captures a genomic DNA target by converting it into a target specific circle using in-solution capture oligonucleo tides [9].

There are limitations with all of the previously described capture methods on genomic DNA from FFPE samples. For example, hybridization enrichment has been applied to cancer samples for single nucleotide variation (SNV) detection [10]. For example, Kerick et al. used the Agilent in-solution hybridization method to investigate reproducibility of SNV detection comparing genomic DNA from FFPE to flash- frozen samples. They demonstrated a false positive rate of approximately 1% when using sequencing coverage greater than 20X coverage. This translates into 1 false mutation caller for every 100 variants identified. In addition, hybridization-based methods have high levels of off-target capture, involve complex workflows that require additional PC amplification and sample preparation steps. MIP technology has potential advantages for degraded genomic DNA from FFPE samples, but the capture reaction is inefficient for larger targets beyond 200bps and the assay is extremely complicated in its implementation [11]. Furthermore, with MIPs, the captured regions contain 20bps of the oligonucleotide-derived sequences and the rest is the reverse- complement of the template DNA, not the original DNA strand. This requires some degree of bioinformatic processing to eliminate synthetic sequence. Capture with the targeted genomic circularization relies on the presence of existing restriction sites in double stranded DNA and requires multiple restriction enzymes which increase the number of reactions needed for a given sample [9]. This can limit the efficiency of capture coverage due to the absence of a suitable restriction site. Furthermore, TGC-capture requires double stranded DNA for restriction enzyme fragmentation while FFPE-derived genomic DNA is generally single stranded. Whole genome amplification using random primers followed by an end- repair step can be used to sequence FFPE-derived genomic DNA, but these amplification steps can skew the representation of certain region even before the capture reaction.

SPECIFIC PATENTS AND PUBLICATIONS

Dahl et al., "Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments," Nucleic Acids Res. 33 e71 (2005), discloses a method for multiplex amplification which uses a general primer pair motif and a vector oligonucleotide selector probe, where the circularization procedure starts with digestion of the DNA to generate targets.

US patent publication 2008/0199916, by Zheng et al., published August 21, 2008, entitled "Multiplex targeted amplification using flap nuclease," discloses the use of UDG (uracil-DNA glycosylase) and a flap exonuclease.

PG Pub 2007/0128635 by Macevicz, entitled "Selected Amplification of

Polynucleotides," discloses a method in which fragments and selection oligonucleotides are combined in a reaction mixture comprising the following enzymatic activities: (i) a 5' flap endonuclease activity, (ii) a DNA polymerase lacking strand displacement activity, (iii) a 3' single stranded exonuclease activity, and (iv) a ligase activity.

WO 2008/033442 A2, "Methods And Compositions For Performing Low Background Multiplex Nucleic Acid Amplification Reactions," by Fredriksson et al., discloses a method of amplifying target nucleic acids involving circularizing target amplicons in an amplified composition; and selecting for said circularized target amplicons in said amplified composition.

BRIEF SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features and aspects of the present invention, nor does it imply that the invention must include all features and aspects discussed in this summary.

The present invention comprises, in certain aspects, methods and materials for detection and analysis of a large number of random fragments of DNA in a sample. The methods can be used for targeted resequencing of DNA. In certain aspects, the present methods employ a mixture of single-stranded polynucleotide capture probes, a number of universal single stranded oligonucleotides (second polynucleotides) each having the same sequence and hybridizing to a portion of the various capture probes; and a mixture comprising exonucleases and a ligase. In certain aspects, the present invention comprises a composition in the form of a reaction mixture useful for preparing a population of double stranded DNA molecules from a sample containing single stranded polynucleic acids, comprising, preferably in a suitable buffer: (a) a plurality of single stranded capture probes, each capture probe containing (i) 5' and 3' end capture arms complementary to specific portions of a polynucleic acid in the sample and (ii) an invariant sequence between the capture arms, whereby a circular structure comprising a specific capture probe and a polynucleic acid sample molecule having regions complementary to the capture arms is formed in the buffer; (b) a plurality of second

("universal") single stranded polynucleotides having a sequence complementary to the invariant sequence and having amplification sites for amplification of a polynucleic acid in a circular structure; and (c) a 5' exonuclease, a 3' exonuclease, and a ligase. While "each" capture probe will contain the defined features, it is not to be implied that "every" capture probe in a composition must have these features. The single stranded polynucleic acids in the composition may comprise random fragments of human genomic DNA. The fragments may be fixed by crosslinking and embedded in a wax, which makes the composition well suited for dealing with degraded DNA from FFPE samples. The composition also comprises at least one of amplification primers and a polymerase for amplification. The amplification sites of the composition comprise PGR primer sites, which may be spaced on the universal polynucleotides about 120 to 250 bases apart.

In certain embodiments, the composition (reaction mixture) comprises capture probes having a three part construction: two capture arms on the flanks which are able to capture specific single-stranded genomic DNA and a sequence between the two capture arms which is termed a "Universal" sequence in that it is essentially the same ("invariant") among the different probes. The capture probes may be present in the composition as a set of at least 500 different probes, at least 600 different probes, at least 700 different probes, or at least 1000 different probes, each probe having capture arms complementary to different portions of a single stranded polynucleic acid in the sample and having the same universal probe sequence between the two capture arms.

In certain aspects, the present invention also comprises a method for analyzing single stranded polynucleotides from a sample, comprising the steps of: (a) adding to the sample a plurality of capture probes, each capture probe containing capture arms designed to be complementary to specific portions of a polynucleic acid in the sample and a universal probe sequence between the arms, whereby a circular structure comprising a specific capture probe and a polynucleic acid sample molecule is formed in the buffer; (b) adding to the sample a plurality of universal polynucleotides having a sequence complementary to the universal probe sequence and having amplification sites for amplification of a polynucleic acid in a circular structure; and (c) adding to the sample containing capture probes and universal polynucleotides a mixture of a 5 ' exonuclease, a 3 ' exonuclease, and a ligase under conditions whereby exonucleases remove bases from the single stranded polynucleotides to form a new 5' end thereof and a new 3' end thereof, and the ligase ligates the new 5' end to the new 3 ' end.

The composition and method described above may also comprise a 5' exonuclease, which may be Exonuclease I; a 3' exonuclease, which may be a polymerase or a thermostable polymerase; and a ligase, which may be a thermostable DNA ligase. As described below, the capture arms may hybridize to various portions of the DNA in the sample, leaving "flaps", which are removed by the exonucleases.

In certain aspects, the present invention further contemplates a method for analyzing single stranded polynucleotides from a sample, comprising the steps of: (a) adding to the sample a plurality of capture probes, each capture probe containing capture arms complementary to specific portions of a polynucleic acid in the sample and a universal probe sequence between the arms, whereby a circular structure comprising a specific capture probe and a polynucleic acid sample molecule is formed in the buffer; (b) adding to the sample a plurality of universal polynucleotides having a sequence complementary to the universal probe sequence and having amplification sites for amplification of a polynucleic acid in a circular structure; and (c) adding to the sample containing capture probes and universal polynucleotides a mixture of a 5' exonuclease, a 3' exonuclease, and a ligase under conditions whereby exonucleases remove bases from the single stranded polynucleotides to form a new 5' end thereof and a new 3' end thereof, and the ligase ligates the new 5' end to the new 3' end; (d) adding to the sample a polymerase and polymerase primers; and (e) conducting a polymerase chain reaction using the polymerase primers for amplification of a portion of a single stranded polynucleotide captured by a corresponding capture probe.

The above method may further comprise the step of sequencing amplified polynucleotides from step (e). The polymerase chain reaction conducted step (e) may utilize an annealing temperature of between about 45 degrees Celsius and 55 degrees Celsius.

The analyzing of the single stranded polynucleotides from a sample may comprise analyzing polynucleotides from a preserved tissue sample or analyzing polynucleotides from a preserved tissue sample and analyzing polynucleotides from a fresh sample from the same individual.

In certain aspects, the present invention also comprises the preparation of a composition as described herein using a kit. The kit may comprise a set of capture probes and universal oligos. Other reagents, such as enzymes may also be included in the kit. An exemplary set of 628 capture polynucleotides is described in the accompanying sequence listing.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A, IB is a schematic diagram illustrating an overview of the single stranded DNA capture assay. Figure 2A, 2B, and 2C is a set of graphs showing the sequencing coverage of targeted resequencing on matched FFPE versus flash- frozen genomic DNA sources in exemplary patients 751 (Fig. 2A), patient 761 (Fig. 2B) and patient 780 (Fig. 2C). Coverage exceeded 85% of all captured regions in each case. Figure 3 is a scatter plot showing where the 2^nd base frequency of a given variant is compared from targeted resequencing of genomic DNA from matched flash- frozen versus FFPE samples. The x-axis represents the 2^nd base frequency of SNVs identified from FFPE targeted resequencing compared to the y-axis, which indicates the variant base fraction from the flash- frozen genomic DNA.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

OVERVIEW

Described herein is a novel DNA targeting and enrichment method particularly suited for analysis of samples containing fragmented single stranded nucleic acids, such as genomic DNA fragments in a biopsy sample. The method results in highly multiplexed amplification of selected portions of the sample nucleic acid, i.e., the reaction mixture may contain hundreds or thousands of different capture probes for amplification of sample DNA regions spanned by the capture probes. The amplified portions from the reaction may be further analyzed, e.g. by sequencing the amplified portions.

The present method is an improvement of a previously described technique that required double stranded DNA as input and required that the targeting oligonucleotide probes be placed adjacent to certain restriction sites. For the present approach, the hybridization arms of the capture oligonucleotides do not require a restriction site and the input DNA can be single stranded. This improves the flexibility and the coverage of the design. An important feature of the present capture approach involves using single stranded DNA as input material. Given the need for high heat during processing, the majority of formalin fixed and paraffin embedded (FFPE) derived genomic DNA molecules are generally single stranded. The present approach has a major advantage compared to other methods that rely exclusively on enzymatic manipulations of double stranded genomic DNA. The capture performance is comparable when using genomic DNA derived from flash- frozen versus FFPE processed tissue. Eighty five percent of the heterozygote SNV detected from high quality genomic DNA extracted flash- frozen samples were also detected in targeted resequencing data from the matched FFPE samples. The number of false positive FFPE- specific SNV calls are exceptionally low at one per every 12 Kb of targeted genomic sequence.

While multiplexed capture assays for hundreds of genomic regions in the present examples is described herein, it is believed the reaction could be scaled to thousands. As published, efficient capture using pools of 5,000 oligonucleotides for restriction enzyme- based targeted circularization has been achieved and it is believed that this new method will scale similarly. For most of the results presented here, we used 4 indexed samples per lane of sequencing (2 flash frozen and 2 FFPE samples). Targeted resequencing projects involving hundreds of exons in hundreds of FFPE samples are therefore achievable and may be implemented with minimal additional steps in a next generation sequencer such as the Illumina HiSeq or GAIIx. In addition, the application of the present approach is demonstrated using the Illumina MiSeq system which is designed for rapid analysis.

An innovative approach to capture genomic targets from archival genomic DNA with in-solution polynucleotides is described. This approach is fundamentally different than other methods given that it only requires random fragments of single stranded genomic DNA as commonly seen in FFPE samples, is highly scalable for multiplexed target coverage, and does not rely on any whole genome amplification. The capture assay is straightforward, relatively fast and can be implemented with standard molecular biology equipment. The robust performance of the capture assay and comparisons of SNV detection using genomic DNA derived from matched flash- frozen and FFPE samples is demonstrated.

The technology described utilizes oligonucleotide-mediated genomic capture without the need for double stranded template and the reliance on exiting restriction sites. It also alleviates the need to synthesize the complementary stranded of the template DNA, which can result in significant limits such as the target size.

Another novel aspect of this capture process is its ability to add desired sequences (such as the adapter sequences required for cluster generation on the Illumina® sequencing system) to DNA fragments without the need for the multi-step process normally associated with such manipulation. This can greatly simplify and accelerate the construction of sequencing libraries. That is, the original

Figure 1A and IB outline the key materials, intermediates and steps of the capture reaction. As shown in these figures, a number of capture probes 101 and a sample containing numerous fragments of single stranded DNA 102 are mixed in a single tube (Step 1). The term "tube" is used for convenience, in that the reaction area could also be a well in a microtiter plate, a chamber in a microfluidic device, etc. The entire reaction occurs in the single tube and this substantially reduces the complexity of the capture assay process. The capture probes and single stranded DNA fragments are mixed in the presence of Ampligase, TaqPol, and Exol. The capture probes 101 have capture arms that are different in sequence as between capture probes and are complementary to the ends of the portion of sample DNA 102 to be studied.

Denatured single-stranded genomic DNA 102 having a 5' end and a 3' end is combined with a pool of polynucleotides, termed "capture probes," that mediate targeted circularization of the regions of interest. Since the size of DNA 102 is unknown and variable ("random"), portions of the DNA 102 will extend 5' and 3' from the hybridization sites, as shown in step 1. The capture probes are single stranded DNA molecules that may be e.g. 80 bases long, or in the range of 40 to 300 bases long. A single capture probe will have 5' capture arm 104, a middle portion 105 ("universal probe sequence") and a 3' capture arm 106 (Fig. IB). The capture arms 104, 106 are typically on the order of 20 bases long, and have a sequence selected for an individual capture probe to target a pre-determined complementary region on the nucleic acid sample. This complementarity is designed to be 100%

complementarity. The region targeted will typically be longer than the capture probe; it may, for example, be an exon of a gene. The middle portion 105 of the capture probe ("universal probe sequence") is selected to have a sequence that will not hybridize to the nucleic acid sample, and its length is chosen depending on the size of the region of the sample (e.g.

genomic DNA) being targeted, and in accordance with the size of the universal oligo. While there are many different capture probe sequences, the middle portion of each capture probe will be essentially the same in each capture probe, in order to hybridize to the universal polynucleotides, as explained below.

Genomic DNA in the sample can come from either flash- frozen or FFPE processed tissue samples. Each capture arm 104, 106 from a single capture probe anneals to a predetermined sequence in a specific genomic DNA fragment 102 containing the

complementary sequences. After hybridization, a single- stranded target-specific structure is formed which has 5' single stranded extension 111 and 3' single stranded extension 112 of the original genomic target single stranded DNA (Fig. IB). These extensions 111, 112 of single stranded genomic DNA (that extend past the ends of the targeting arms of the capture probe) are removed or degraded by enzymes. For example 5' and 3' extensions ("flaps") may be removed, respectively, by the 5' nucleo lytic activity of Taq polymerase (activity as disclosed, e.g. in Lyamichev, V., Brow, M. A. & Dahlberg, J. E. (1993) Science 260, 778- 783) and the 3' to 5' exonucleolytic activity of Exol [12]. To complete the capture reaction, a universal vector oligonucleotide 108 anneals to the general sequence motif in the middle portion 105 of every capture probe oligonucleotide. Ampligase® thermostable ligase present in the same reaction mix forms covalently closed circles using the universal vector sequence (Step 2). Ampligase® Thermostable DNA Ligase catalyzes NAD-dependent ligation of adjacent 3 '-hydroxylated and 5'-phosphorylated termini in duplex DNA structures that are stable at high temperatures.

Once the circle is complete, universal PC primers 110 can be used to amplify the intervening target genomic DNA fragment, creating a pool of linear amplicons that can be sequenced (Step 3). The primers are oriented, as shown in Figure IB, to amplify the target oligonucleotide; they can be amplified either as an intact circle, or after cleavage of the circle. The resulting double stranded linear DNA population that results from amplification of the set of circles created is then submitted to adapter ligation following the standard Illumina library preparation protocol (Step 4). The primers hybridize to sequences within the universal sequences, so that one set of primers may be used to amplify the entire plurality of different capture probe structures. As shown by arrows 110 in Figure IB, the PCR amplification can proceed from the primers through part of the general sequence motif in the middle portion 105 of the capture probe. This allows sequences from this motif to be added to and become part of the 5' and/or 3' end of the amplified product. For example, bar codes or ligation adapters can be added by including such sequences in the middle portion 105 of the capture probe. A variety of sequencing methods may be used on the amplified products, including massively parallel methods commercially available from Illumina, Roche 454, Life Technologies, Pacific Biosciences, Helicos, etc. The sequencing aspect of the present methods can be used for SNP analysis as well as SNVs that are associated with disease. The sequencing libraries prepared by the present method can be used for paired-end sequencing to obtain greater information from a ssDNA fragment in the sample.

A variety of buffers can be used with the present compositions. They can contain, e.g. lOOmM Tris-Cl, 500mM KC1; 600mM Tris-Cl, 170mM (NH4)2S04, 0.1% Tween-20;

375mM Tris-Cl, 200mM (NFL^SC , 0.1% Tween-20, etc. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. Generally, nomenclatures utilized in connection with, and techniques of, cell and molecular biology and chemistry are those well-known and commonly used in the art. Certain experimental techniques, not specifically defined, are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. For purposes of clarity, the following terms are defined below.

Ranges: For conciseness, any range set forth is intended to include any sub-range within the stated range, unless otherwise stated. A sub-range is to be included within a range even though no sub-range is explicitly stated in connection with the range. As a nonlimiting example, a range of 120 to 250 includes a range of 120-121, 120-130, 200-225, 121-250 etc. The term "about" has its ordinary meaning of approximately and may be determined in context by experimental variability. In case of doubt, "about" means a variation within 5% of a stated numerical value. The term "polynucleotide" corresponds to either double-stranded or single- stranded cDNA or genomic DNA or RNA, containing at least 10 contiguous nucleotides. Single stranded polynucleic acid sequences are always represented in the current invention from the 5' end to the 3' end. Polynucleic acids according to the invention may be prepared by any method known in the art for preparing polynucleic acids (e.g. the phosphodiester method for synthesizing oligonucleotides as described by Agarwal et al. (1972), the phosphotriester method of Hsiung et al. (1979), or the automated diethylphosphoroamidite method of Baeucage et al. (1981)). Alternatively, the polynucleic acids of the invention may be isolated fragments of naturally occurring or cloned DNA or RNA.

The term "oligonucleotide" refers to a single stranded nucleic acid comprising two or more nucleotides, and less than 300 nucleotides. The exact size of an oligonucleotide depends on the ultimate function or use of said oligonucleotide. For use as a probe or primer the oligonucleotides are preferably about 5-50 nucleotides long. The oligonucleotides and polynucleotides according to the present invention can be formed by cloning of recombinant plasmids containing inserts including the corresponding nucleotide sequences, if need be by cleaving the latter out from the cloned plasmids upon using the adequate nucleases and recovering them, e.g. by fractionation according to molecular weight. The probes according to the present invention can also be synthesized chemically, e.g. by automatic synthesis on commercial instruments sold by a variety of manufacturers.

The nucleotides as used in the present invention may, in certain aspects, be ribonucleotides, deoxyribonucleotides and modified nucleotides such as inosine or nucleotides containing modified groups which do not essentially alter their hybridisation characteristics. Moreover, it is obvious to the man skilled in the art that any of the below- specified probes can be used as such, or in their complementary form, or in their RNA form (wherein T is replaced by U).

The oligonucleotides used as primers or probes may also comprise or consist of nucleotide analogues such as phosphorothioates (Matsukura et al., 1987).

alkylphosphorothioiates (Miller et al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or may contain intercalating agents (Asseline et aL, 1984).

The term "probe" refers to single stranded sequencespecific oligonucleotides which have a sequence which is sufficiently complementary to hybridize to the target sequence to be detected. Preferably said probes are 70%, 80%, 90%, or more than 95% homologous to the exact complement of the target sequence to be detected. These target sequences are either genomic DNA or messenger RNA, or amplified versions thereof. Preferably, these probes are about 5 to 50 nucleotides long, more preferably from about 10 to 30 nucleotides.

The term "hybridizes to" refers to preferably stringent hybridizations conditions, allowing hybridisation between complementary nucleic acid sequences showing at least 90%, 95% or more homology with each other.

The term "primer" refers to a single stranded DNA oligonucleotide sequence capable of acting as a point of initiation for synthesis of a primer extension product which 5 is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products.

Preferably the primer is about 5-50 nucleotides long. Specific length and sequence will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength. The fact that amplification primers do not have to match exactly with the corresponding template sequence to warrant proper amplification is amply documented in the literature. The amplification method used can be either polymerase chain reaction, target polynucleotide amplification methods such as self- sustained sequence replication (3SR) and strand-displacement amplification (SDA); methods based on amplification of a signal attached to the target polynucleotide, such as "branched chain" DNA amplification; methods based on amplification of probe DNA, such as ligase chain reaction (LCR) and QB replicase amplification (QBR); transcription-based methods, such as ligation activated transcription (LAT), nucleic acid sequence-based amplification (NASBA), amplification under the trade name INVADER, and transcription- mediated amplification (TMA); and various other amplification methods, such as repair chain reaction (RCR) and cycling probe reaction (CPR). Preferred methods can be multiplexed, i.e. a number of amplifications of different sequences can be run in the same reaction mixture at the same time. The term "complementary" nucleic acids as used in the current invention means that the nucleic acid sequences can form a perfect base paired double helix with each other.

The term "FFPE" refers to formalin- fixed, paraffin-embedded (FFPE) tissue samples. Commercial solutions of formadehyde in water are commonly called formalin. Formalin preserves or fixes tissue or cells by reversibly cross-linking primary amino groups in proteins with other nearby nitrogen atoms in protein or DNA through a -C¾- linkage.

Tissue samples are typically placed into molds along with liquid embedding material (such as agar, gelatine, or wax) which is then hardened. This is achieved by cooling in the case of paraffin wax and heating (curing) in the case of the epoxy resins. The acrylic resins are polymerised by heat, ultraviolet light, or chemical catalysts. The hardened blocks containing the tissue samples are then ready to be sectioned.

Another aldehyde that can be used for fixation is glutaraldehyde. It operates in a similar way to formaldehyde by causing deformation of the alpha-helix structures in proteins. However, glutaraldehyde is a larger molecule, and so its rate of diffusion across membranes is slower than formaldehyde. Samples that may be used in the present invention include medical samples, forensic samples, museum or archeological samples, and other archival collections, which need not be FFPE preserved. There are many preservation methods that have been applied to tissues, including alcohol preservation, formalin treatment, freezing and sequestration in waxes and other materials. In addition, forensic or archeological samples may contain degraded ssDNA that has not been consciously preserved at all.

The term "5' exonuclease" or "5' end nuclease" refers to an enzyme that has activity 5' to 3' direction to remove a single stranded DNA having a 5' end. It may do this through exonuclease or endonuclease activity, i.e. cleavage at a point where the ssDNA separates from its complementary strand. The 5' exonuclease enzymes used herein preferably degrade single stranded DNA, not double stranded DNA. The preferred 5' exonuclease is a DNA polymerase that has the ability to cleave a DNA hairpin where a 5' end of DNA to be cleaved is a single strand adjacent to a double strand, which may result from formation of an exogenous duplex, such as hybridization to a primer. For details, see Lyamichev et al.

"Structure- Specific Endonucleolytic Cleavage of Nucleic Acids by Eubacterial DNA

Polymerases," Science 260:778-783 (1993), describing this activity in DNAP-Ecl and DNAP-Taq (from Thermus aquaticus) polymerases. The term "3' exonuclease" or "3' end nuclease" refers to an enzyme having activity in the 3' to 5' direction to remove a single stranded DNA portion having a 3' end. As with the 5' exonuclease, the enzyme will only act on ssDNA and may do this by either exonuclease or endonuclease activity. This activity is found as DNA proofreading in certain DNA polymerases. It allows the enzyme to check each nucleotide during DNA synthesis, and excise mismatched nucleotides in the 3 ' to 5 ' direction. The proofreading domain also enables a polymerase to remove unpaired 3 ^' overhanging nucleotides to create blunt ends. Protocols such as high-fidelity PC , 3 ' overhang polishing and high-fidelity second strand synthesis require the presence of a 3 '→ 5 ' exonuclease.

The preferred 3' exonuclease is Exo I. Exonuclease I (Exo I), the product of the sbcB gene of E. coli, is an exodeoxyribonuclease that hydrolyzes single- stranded (ss)DNA stepwise in a 3' to 5' direction. 1-3 Hydrolysis generates deoxyribonucleoside 5'- monophosphates and a terminal dinucleotide diphosphate. The enzyme requires magnesium (optimal Mg++ concentration is 10 mM) and the presence of a free 3'-hydroxyl terminus. Exonuclease I is active under a wide variety of buffer conditions, allowing addition of the enzyme directly into most reaction mixes. Heat inactivation results from incubation at 80° C for 15 minutes.

The term "ligase" refers to an enzyme that catalyzes formation of a phosphodiester bond between the 5' phosphate of one strand of DNA and the 3' hydroxyl of the other. This enzyme is used to covalently link or ligate f agments of DNA together. An example of a DNA ligase is one derived from the T4 bacteriophage. T4 DNA ligase requires ATP as a cofactor. The presently preferred ligase is Ampligase® ligase (registered trademark of Epicentre Technologies), a thermostable DNA ligase that catalyzes NAD-dependent ligation of adjacent 3'-hydroxylated and 5'-phosphorylated termini in duplex DNA structures that are stable at high temperatures.

For convenience, certain polynucleotides are referred to herein as "capture probes," meaning single stranded polynucleotides of relatively small size, e.g. 40-4000 bases, which are prepared (e.g. synthetically) to contain defined features. These include certain "universal" sequences, which are so designated because they are essentially identical as between different polynucleotides designed for the stated purpose, whereas other sequences in the capture probes will vary among a number of different possibilities to capture different targets. That is, the capture probes contain a "universal probe sequence" which contains a single sequence common to all capture probes. In this way, the "universal polynucleotides" may have a single sequence that is complementary to the universal sequence in the capture probes.

EXAMPLES

EXAMPLE 1; Oligonucleotide design, target DNA capture, and sequencing

Samples Genomic DNA from NA18507 was obtained from Corriel Cell Repositories.

Intestinal tissue samples were obtained from under an IRB protocol approved by Stanford University. These samples were either immediately snap frozen in liquid nitrogen and stored at -80°C or preserved as formalin- fixed, paraffin-embedded (FFPE) blocks. Total nucleic acids were extracted from the flash- frozen tissue using the SQ DNA/RNA/Protein Kit from Omega Bio-Tek. Following complete RNase A digestion, the DNA (herein referred as dsDNA) was analyzed by argarose gel electrophoresis and quantified by a fluorescence assay using SYBR Gold (Invitrogen). For FFPE samples, DNA was isolated using the BiOstic® FFPE Tissue DNA Isolation Kit from Mo Bio Laboratories. The quantity and quality of the preparations were by OD260 and qPCR analysis across 3 different genomic loci. Only single stranded DNA (ssDNA) samples with a difference in Ct values of equal or less than 4.0 or approximately 15% genome equivalence between the flash- frozen and FFPE samples were used for subsequent analysis. Capture Polynucleotides and Sequence Listing

Capture polynucleotides with the properties optimal for FFPE capture were chosen from a larger, previously described set (Natsoulis et al. 2011, Ref. 9). As disclosed there, the oligonucleotide sequences can be downloaded from the Human OligoExome, a database which provides gene exons annotated by the Consensus Coding Sequencing Project (CCDS). The database is available at oligoexome.Stanford.edu. 628 capture oligonucleotides resulting in amplicons ranging from 150 to 250bp were chosen from this set. 2,512 sequences containing sequences of the 5' targeting arm, 3' targeting arm, amplicon, and target oligonucleotide for each of the 628 capture oligonucleotides were compiled. Targeting arms were positioned in regions without SNPs per dbSNP. Details on the design parameters and on the capture characteristics of the targeting arms are provided by Natsoulis et al. [9].

The accompanying sequence listing sets forth the sequences of the 5' targeting arm, the 3' targeting arm, the amplicon sequence and the universal oligonucleotide used, including uridine substitutions for the 628 capture probes used in the examples. In the table below, Column 1 is the chromosome number targeted; column 2 is the position of the 5' end of the targeted sequence; col. 3 is the polarity of the targeted strand; column 4 (SEQ ID NOs) is sequence of the 20 bp 5' targeting arm; column 5 (SEQ ID NOs) is the sequence of the 3' 20 bp selector; column 6 (SEQ ID NOs) lists the sequences of the amplicons and column 7 (SEQ ID NOs) lists the sequences of the targeting oligonucleotides ("universal probes") including uridine substitutions; and column 8 is the identifier (which may also be checked at the Stanford OligoExome web site).

61808885 minus 57 58 59 60 ARFRP1 ROI 1

10959598 minus 61 62 63 64 SMARCA4 ROI 5

106813126 minus 65 66 67 68 S 6GAL2 ROI 4

65266899 minus 69 70 71 72 SMAD3 ROI 8

51168635 minus 73 74 75 76 TCF4 ROI 7

50666736 minus 77 78 79 80 ACVR1B ROI 7

89472909 minus 81 82 83 84 EPHA3 ROI 4

69635044 minus 85 86 87 88 DLG3 ROI 17

148157809 minus 89 90 91 92 EZH2 ROI 4

98391555 plus 93 94 95 96 TRRAP ROI 37

113461681 minus 97 98 99 100 CSMD3 ROI 39

55296258 plus 101 102 103 104 PCDH15 ROI 26

1778491 plus 105 106 107 108 FGFR3 ROI 12

6117503 minus 109 110 111 112 CHD5 ROI 16

28400927 minus 113 114 115 116 NF2 ROI 13

85666939 plus 117 118 119 120 EED ROI 12

10982204 minus 121 122 123 124 SMARCA4 ROI 14

112129928 minus 125 126 127 128 APC ROI 2

65269671 minus 129 130 131 132 SMAD3 ROI 9

11115574 plus 133 134 135 136 FRAP1 ROI 35

35000025 minus 137 138 139 140 CCNE1 ROI 4

35461947 minus 141 142 143 144 SRC ROI 7

107633539 minus 145 146 147 148 ATM ROI 13

173603051 minus 149 150 151 152 TNR ROI 8

6093131 minus 153 154 155 156 CHD5 ROI 34

140124110 plus 157 158 159 160 BRAF ROI 12

46838465 minus 161 162 163 164 SMAD4 ROI 5

85954365 plus 165 166 167 168 DACH2 ROI 8

132282116 minus 169 170 171 172 NEK11 ROI 2

69638588 plus 173 174 175 176 DLG3 ROI 22

20487399 minus 177 178 179 180 CYFIP1 ROI 6

20479945 minus 181 182 183 184 CYFIP1 ROI 3

80806019 minus 185 186 187 188 TTK ROI 17

197966150 minus 189 190 191 192 SF3B1 ROI 21

77093105 minus 193 194 195 196 NAV3 ROI 25

55260662 minus 197 198 199 200 KIT ROI 4

11110373 minus 201 202 203 204 FRAP1 ROI 41

122364376 plus 205 206 207 208 GRIA3 ROI 8

113771268 minus 209 210 211 212 CSMD3 ROI 15

89604424 minus 213 214 215 216 EPHA3 ROI 16

179352082 minus 217 218 219 220 TTN ROI 22

24524011 minus 221 222 223 224 CDH10 ROI 11

64331720 minus 225 226 227 228 MEN1 ROI 3

11013228 minus 229 230 231 232 SMARCA4 ROI 29

69585530 minus 233 234 235 236 DLG3 ROI 2

107619747 minus 237 238 239 240 ATM ROI 4

74782245 minus 241 242 243 244 TNNI3K ROI 23

42922016 minus 245 246 247 248 RET ROI 5

79990425 minus 249 250 251 252 CTNNA2 ROI 6

197978280 plus 253 254 255 256 SF3B1 ROI 10

89714874 plus 257 258 259 260 PTEN ROI 9

55234021 minus 261 262 263 264 EGFR ROI 24

23629188 plus 265 266 267 268 ERN2 ROI 3

20542653 minus 269 270 271 272 CYFIP1 ROI 22

41921446 minus 273 274 275 276 ATP5A1 ROI 6

69590881 minus 277 278 279 280 DLG3 ROI 10

24628976 plus 281 282 283 284 CDH10 ROI 1

49086077 minus 285 286 287 288 DCC ROI 13

10999310 plus 289 290 291 292 SMARCA4 ROI 23

10377190 minus 293 294 295 296 MYH2 ROI 13 179372871 minus 297 298 299 300 TTN ROI 4

31804798 plus 301 302 303 304 BRCA2 ROI 8

151467266 minus 305 306 307 308 MLL3 ROI 56

173559224 minus 309 310 311 312 TNR ROI 21

32189350 plus 313 314 315 316 FHOD3 ROI 2

55332859 plus 317 318 319 320 PCDH15 ROI 25

107723290 minus 321 322 323 324 ATM ROI 56

113392559 minus 325 326 327 328 CSMD3 ROI 51

37809872 minus 329 330 331 332 GPR124 ROI 9

10993251 plus 333 334 335 336 SMARCA4 ROI 18

47309470 minus 337 338 339 340 ARAF ROI 3

31810986 plus 341 342 343 344 BRCA2 ROI 9

77039778 minus 345 346 347 348 NAV3 ROI 16

130056498 minus 349 350 351 352 GPR133 ROI 11

197980963 minus 353 354 355 356 SF3B1 ROI 9

3056175 minus 357 358 359 360 RIPK1 ROI 9

119918586 minus 361 362 363 364 HNF1A ROI 5

81479256 plus 365 366 367 368 CACNA2D1 ROI 15

85856366 minus 369 370 371 372 DACH2 ROI 6

35461782 plus 373 374 375 376 SRC ROI 7

7518320 minus 377 378 379 380 TP53 ROI 5

35464392 minus 381 382 383 384 SRC ROI 9

148157603 plus 385 386 387 388 EZH2 ROI 4

113346109 minus 389 390 391 392 PPP1R3A ROI 1

55286630 plus 393 394 395 396 KIT ROI 9

55257300 minus 397 398 399 400 PCDH15 ROI 31

6092590 minus 401 402 403 404 CHD5 ROI 35

1405894 minus 405 406 407 408 TPO ROI 2

31842389 plus 409 410 411 412 BRCA2 ROI 17

173639039 minus 413 414 415 416 TNR ROI 2

20896668 minus 417 418 419 420 ZNF521 ROI 7

81533723 minus 421 422 423 424 CACNA2D1 ROI 8

1777450 minus 425 426 427 428 FGFR3 ROI 8

173598272 plus 429 430 431 432 TNR ROI 12

112182432 plus 433 434 435 436 APC ROI 9

29589493 plus 437 438 439 440 HM13 ROI 3

74569805 minus 441 442 443 444 TNNI3K ROI 6

138672620 minus 445 446 447 448 PCDH18 ROI 1

180405054 minus 449 450 451 452 PIK3CA ROI 5

80778400 minus 453 454 455 456 TTK ROI 6

130037831 minus 457 458 459 460 GPR133 ROI 5

179332128 minus 461 462 463 464 TTN ROI 37

3028510 minus 465 466 467 468 RIPK1 ROI 4

113881663 minus 469 470 471 472 CSMD3 ROI 14

98327833 plus 473 474 475 476 TRRAP ROI 4

29566181 minus 477 478 479 480 HM13 ROI 1

113368401 minus 481 482 483 484 CSMD3 ROI 59

1002034 minus 485 486 487 488 ABCA7 ROI 14

122366090 plus 489 490 491 492 GRIA3 ROI 10

31798088 plus 493 494 495 496 BRCA2 ROI 4

10967739 plus 497 498 499 500 SMARCA4 ROI 9

20487151 plus 501 502 503 504 CYFIP1 ROI 6

122426247 plus 505 506 507 508 GRIA3 ROI 13

20512312 minus 509 510 511 512 CYFIP1 ROI 15

107633350 minus 513 514 515 516 ATM ROI 13

49874078 plus 517 518 519 520 CAMKV ROI 3

35134965 minus 521 522 523 524 ERBB2 ROI 18

173615171 minus 525 526 527 528 TNR ROI 7

122215055 minus 529 530 531 532 GRIA3 ROI 3

998634 minus 533 534 535 536 ABCA7 ROI 11 10990483 plus 537 538 539 540 SMARCA4 ROI 16

49102265 plus 541 542 543 544 DCC ROI 14

14540423 plus 545 546 547 548 TRIO ROI 47

3022200 minus 549 550 551 552 RIPK1 ROI 1

55260490 plus 553 554 555 556 KIT ROI 4

98371213 minus 557 558 559 560 TRRAP ROI 26

70138988 plus 561 562 563 564 BAI3 ROI 28

47863727 plus 565 566 567 568 MSH6 ROI 1

20542423 plus 569 570 571 572 CYFIP1 ROI 22

35465175 minus 573 574 575 576 SRC ROI 11

11030314 plus 577 578 579 580 SMARCA4 ROI 31

76939508 plus 581 582 583 584 NAV3 ROI 9

179374960 plus 585 586 587 588 TTN ROI 2

10375909 minus 589 590 591 592 MYH2 ROI 14

14452158 minus 593 594 595 596 TRIO ROI 29

32410317 minus 597 598 599 600 FHOD3 ROI 6

132430096 minus 601 602 603 604 NEK11 ROI 12

113654983 minus 605 606 607 608 CSMD3 ROI 25

98440747 minus 609 610 611 612 TRRAP ROI 63

3030397 minus 613 614 615 616 RIPK1 ROI 5

1008764 plus 617 618 619 620 ABCA7 ROI 27

26687327 plus 621 622 623 624 NF1 ROI 41

69582111 minus 625 626 627 628 DLG3 ROI 1

151480573 plus 629 630 631 632 MLL3 ROI 48

58744170 plus 633 634 635 636 OMA1 ROI 6

113306238 plus 637 638 639 640 CSMD3 ROI 72

26688035 minus 641 642 643 644 NF1 ROI 42

24545302 plus 645 646 647 648 CDH10 ROI 6

10325887 minus 649 650 651 652 TYK2 ROI 13

41663548 minus 653 654 655 656 FOXP4 ROI 7

6092956 plus 657 658 659 660 CHD5 ROI 34

70600532 minus 661 662 663 664 TAF1 ROI 36

6089133 minus 665 666 667 668 CHD5 ROI 37

51087922 minus 669 670 671 672 TCF4 ROI 10

173598457 plus 673 674 675 676 TNR ROI 12

20549735 plus 677 678 679 680 CYFIP1 ROI 25

1004178 plus 681 682 683 684 ABCA7 ROI 18

41663374 minus 685 686 687 688 FOXP4 ROI 7

28384229 minus 689 690 691 692 NF2 ROI 7

113335653 minus 693 694 695 696 CSMD3 ROI 64

6110543 plus 697 698 699 700 CHD5 ROI 22

114457993 plus 701 702 703 704 CSMD3 ROI 2

26532904 minus 705 706 707 708 NF1 ROI 7

107626580 plus 709 710 711 712 ATM ROI 8

113598298 minus 713 714 715 716 CSMD3 ROI 29

73535839 plus 717 718 719 720 PDZRN3 ROI 4

130056320 plus 721 722 723 724 GPR133 ROI 11

102504422 minus 725 726 727 728 CDC42BPB ROI 15

55370482 plus 729 730 731 732 PCDH15 ROI 23

85638836 plus 733 734 735 736 EED ROI 2

67406769 plus 737 738 739 740 CDH1 ROI 10

14431209 minus 741 742 743 744 TRIO ROI 20

179365142 minus 745 746 747 748 TTN ROI 8

179377374 plus 749 750 751 752 TTN ROI 1

130186634 minus 753 754 755 756 GPR133 ROI 21

179363060 minus 757 758 759 760 TTN ROI 10

1771021 minus 761 762 763 764 FGFR3 ROI 2

80669895 minus 765 766 767 768 CTNNA2 ROI 14

113307388 minus 769 770 771 772 PPP1R3A ROI 3

70246581 plus 773 774 775 776 IL2RG ROI 4 10325294 minus 111 778 779 780 TYK2 ROI 14

119911190 minus 781 782 783 784 HNF1A ROI 2

48121006 plus 785 786 787 788 DCC ROI 1

112144471 minus 789 790 791 792 APC ROI 5

6106725 minus 793 794 795 796 CHD5 ROI 28

107373641 minus 797 798 799 800 MGC16169 ROI 14

102480548 minus 801 802 803 804 CDC42BPB ROI 29

179347763 plus 805 806 807 808 TTN ROI 26

69741756 minus 809 810 811 812 BAI3 ROI 8

58777086 minus 813 814 815 816 OMA1 ROI 1

6125223 minus 817 818 819 820 CHD5 ROI 13

114458170 minus 821 822 823 824 CSMD3 ROI 2

130004814 plus 825 826 827 828 GPR133 ROI 1

113771425 minus 829 830 831 832 CSMD3 ROI 15

10991347 minus 833 834 835 836 SMARCA4 ROI 17

10984475 plus 837 838 839 840 SMARCA4 ROI 15

106789533 minus 841 842 843 844 ST6GAL2 ROI 5

10956019 minus 845 846 847 848 SMARCA4 ROI 1

85665798 minus 849 850 851 852 EED ROI 10

3055886 plus 853 854 855 856 RIPK1 ROI 9

25253986 minus 857 858 859 860 KRAS ROI 5

93528815 minus 861 862 863 864 ROR2 ROI 8

64415549 plus 865 866 867 868 ROR1 ROI 9

98412584 minus 869 870 871 872 TRRAP ROI 50

3028223 minus 873 874 875 876 RIPK1 ROI 4

35134678 plus 877 878 879 880 ERBB2 ROI 18

132429912 plus 881 882 883 884 NEK11 ROI 12

20506689 minus 885 886 887 888 CYFIP1 ROI 12

107708549 plus 889 890 891 892 ATM ROI 50

41664415 minus 893 894 895 896 FOXP4 ROI 8

119921304 plus 897 898 899 900 HNF1A ROI 8

32578121 minus 901 902 903 904 FHOD3 ROI 20

107701845 plus 905 906 907 908 ATM ROI 44

14422333 plus 909 910 911 912 TRIO ROI 18

98345465 plus 913 914 915 916 TRRAP ROI 14

20498252 plus 917 918 919 920 CYFIP1 ROI 10

20479614 minus 921 922 923 924 CYFIP1 ROI 2

20492019 plus 925 926 927 928 CYFIP1 ROI 8

20554394 minus 929 930 931 932 CYFIP1 ROI 28

25269940 minus 933 934 935 936 KRAS ROI 3

49874248 minus 937 938 939 940 CAMKV ROI 3

64397295 minus 941 942 943 944 ROR1 ROI 8

10379373 minus 945 946 947 948 MYH2 ROI 12

151486731 plus 949 950 951 952 MLL3 ROI 43

23628844 plus 953 954 955 956 ERN2 ROI 4

26604010 minus 957 958 959 960 NF1 ROI 31

70518168 plus 961 962 963 964 TAF1 ROI 9

151533118 minus 965 966 967 968 MLL3 ROI 25

114360074 minus 969 970 971 972 CSMD3 ROI 4

41252069 plus 973 974 975 976 CTNNB1 ROI 8

42924641 minus 977 978 979 980 RET ROI 6

74473574 plus 981 982 983 984 TNNI3K ROI 1

26681290 plus 985 986 987 988 NF1 ROI 39

14560333 minus 989 990 991 992 TRIO ROI 55

113385976 plus 993 994 995 996 CSMD3 ROI 53

113940635 minus 997 998 999 1000 CSMD3 ROI 12

42943416 plus 1001 1002 1003 1004 RET ROI 20

35463459 minus 1005 1006 1007 1008 SRC ROI 8

64288796 plus 1009 1010 1011 1012 ROR1 ROI 4

107646851 plus 1013 1014 1015 1016 ATM ROI 17 41664210 plus 1017 1018 1019 1020 FOXP4 ROI 8

77108036 plus 1021 1022 1023 1024 NAV3 ROI 33

23629030 minus 1025 1026 1027 1028 ERN2 ROI 4

107695741 plus 1029 1030 1031 1032 ATM ROI 41

98316772 minus 1033 1034 1035 1036 TRRAP ROI 1

28381453 plus 1037 1038 1039 1040 NF2 ROI 6

6114357 minus 1041 1042 1043 1044 CHD5 ROI 18

49177776 minus 1045 1046 1047 1048 DCC ROI 18

35463269 plus 1049 1050 1051 1052 SRC ROI 8

80728470 minus 1053 1054 1055 1056 CTNNA2 ROI 17

151499189 minus 1057 1058 1059 1060 MLL3 ROI 39

3023129 minus 1061 1062 1063 1064 RIPK1 ROI 2

41241299 plus 1065 1066 1067 1068 CTNNB1 ROI 3

10324452 plus 1069 1070 1071 1072 TYK2 ROI 15

11097989 plus 1073 1074 1075 1076 FRAP1 ROI 48

55391619 minus 1077 1078 1079 1080 PCDH15 ROI 21

69842612 minus 1081 1082 1083 1084 BAI3 ROI 15

26686136 minus 1085 1086 1087 1088 NF1 ROI 40

1014393 plus 1089 1090 1091 1092 ABCA7 ROI 31

76886518 minus 1093 1094 1095 1096 NAV3 ROI 5

107376846 plus 1097 1098 1099 1100 MGC16169 ROI 11

140147531 plus 1101 1102 1103 1104 BRAF ROI 6

69585338 plus 1105 1106 1107 1108 DLG3 ROI 2

61808645 plus 1109 1110 1111 1112 ARFRP1 ROI 1

29596459 minus 1113 1114 1115 1116 HM13 ROI 4

31811217 plus 1117 1118 1119 1120 BRCA2 ROI 9

26708193 minus 1121 1122 1123 1124 NF1 ROI 53

64397102 plus 1125 1126 1127 1128 ROR1 ROI 8

20923277 plus 1129 1130 1131 1132 ZNF521 ROI 6

130168789 minus 1133 1134 1135 1136 GPR133 ROI 18

46858794 minus 1137 1138 1139 1140 SMAD4 ROI 10

148160509 plus 1141 1142 1143 1144 EZH2 ROI 3

10334291 minus 1145 1146 1147 1148 TYK2 ROI 6

130186385 plus 1149 1150 1151 1152 GPR133 ROI 21

28362823 minus 1153 1154 1155 1156 NF2 ROI 2

20479420 plus 1157 1158 1159 1160 CYFIP1 ROI 2

151511132 minus 1161 1162 1163 1164 MLL3 ROI 34

11230696 minus 1165 1166 1167 1168 FRAP1 ROI 6

113306026 plus 1169 1170 1171 1172 CSMD3 ROI 72

119918298 plus 1173 1174 1175 1176 HNF1A ROI 5

31810046 minus 1177 1178 1179 1180 BRCA2 ROI 9

24527639 minus 1181 1182 1183 1184 CDH10 ROI 10

26700268 minus 1185 1186 1187 1188 NF1 ROI 49

26709723 minus 1189 1190 1191 1192 NF1 ROI 55

130187321 plus 1193 1194 1195 1196 GPR133 ROI 22

42921680 plus 1197 1198 1199 1200 RET ROI 5

98440554 minus 1201 1202 1203 1204 TRRAP ROI 63

67404719 minus 1205 1206 1207 1208 CDH1 ROI 9

130022035 minus 1209 1210 1211 1212 GPR133 ROI 3

85655898 minus 1213 1214 1215 1216 DACH2 ROI 3

31791419 minus 1217 1218 1219 1220 BRCA2 ROI 2

74607813 minus 1221 1222 1223 1224 TNNI3K ROI 14

20544316 plus 1225 1226 1227 1228 CYFIP1 ROI 23

11029801 plus 1229 1230 1231 1232 SMARCA4 ROI 30

115052754 minus 1233 1234 1235 1236 NRAS ROI 4

132363792 plus 1237 1238 1239 1240 NEK11 ROI 8

49267015 plus 1241 1242 1243 1244 DCC ROI 26

11109744 minus 1245 1246 1247 1248 FRAP1 ROI 42

26506977 plus 1249 1250 1251 1252 NF1 ROI 2

112203353 minus 1253 1254 1255 1256 APC ROI 15 107707231 plus 1257 1258 1259 1260 ATM ROI 48

98417178 plus 1261 1262 1263 1264 TRRAP ROI 53

32515347 minus 1265 1266 1267 1268 FHOD3 ROI 12

107695930 minus 1269 1270 1271 1272 ATM ROI 41

10990654 minus 1273 1274 1275 1276 SMARCA4 ROI 16

179356542 plus 1277 1278 1279 1280 TTN ROI 15

11239670 plus 1281 1282 1283 1284 FRAP1 ROI 3

140100425 plus 1285 1286 1287 1288 BRAF ROI 14

57318669 minus 1289 1290 1291 1292 CDH20 ROI 3

29596261 plus 1293 1294 1295 1296 HM13 ROI 4

86302192 plus 1297 1298 1299 1300 KLHDC4 ROI 9

11127352 minus 1301 1302 1303 1304 FRAP1 ROI 32

20551387 plus 1305 1306 1307 1308 CYFIP1 ROI 27

81450528 minus 1309 1310 1311 1312 CACNA2D1 ROI 23

20496331 plus 1313 1314 1315 1316 CYFIP1 ROI 9

81531406 plus 1317 1318 1319 1320 CACNA2D1 ROI 9

69165113 plus 1321 1322 1323 1324 CCND1 ROI 1

179377566 minus 1325 1326 1327 1328 TTN ROI 1

113305967 plus 1329 1330 1331 1332 PPP1R3A ROI 3

115060096 plus 1333 1334 1335 1336 NRAS ROI 1

119915696 plus 1337 1338 1339 1340 HNF1A ROI 3

80670072 minus 1341 1342 1343 1344 CTNNA2 ROI 14

148175057 plus 1345 1346 1347 1348 EZH2 ROI 1

28384028 plus 1349 1350 1351 1352 NF2 ROI 7

85639014 minus 1353 1354 1355 1356 EED ROI 2

14534677 minus 1357 1358 1359 1360 TRIO ROI 44

132551250 minus 1361 1362 1363 1364 NEK11 ROI 15

148154607 minus 1365 1366 1367 1368 EZH2 ROI 7

20551586 minus 1369 1370 1371 1372 CYFIP1 ROI 27

7882884 plus 1373 1374 1375 1376 MAP2K7 ROI 8

179350505 minus 1377 1378 1379 1380 TTN ROI 24

151510170 minus 1381 1382 1383 1384 MLL3 ROI 35

20477147 minus 1385 1386 1387 1388 CYFIP1 ROI 1

37810214 plus 1389 1390 1391 1392 GPR124 ROI 10

89544938 minus 1393 1394 1395 1396 EPHA3 ROI 10

32428598 plus 1397 1398 1399 1400 FHOD3 ROI 7

1773252 minus 1401 1402 1403 1404 FGFR3 ROI 4

10991137 plus 1405 1406 1407 1408 SMARCA4 ROI 17

14534193 plus 1409 1410 1411 1412 TRIO ROI 43

1777921 plus 1413 1414 1415 1416 FGFR3 ROI 10

107435699 minus 1417 1418 1419 1420 MGC16169 ROI 2

21958177 plus 1421 1422 1423 1424 CDKN2A ROI 4

113315829 minus 1425 1426 1427 1428 CSMD3 ROI 69

36755101 minus 1429 1430 1431 1432 DCLK3 ROI 1

102539967 plus 1433 1434 1435 1436 CDC42BPB ROI 4

49102472 minus 1437 1438 1439 1440 DCC ROI 14

64330363 minus 1441 1442 1443 1444 MEN1 ROI 5

74606037 minus 1445 1446 1447 1448 TNNI3K ROI 12

115052549 plus 1449 1450 1451 1452 NRAS ROI 4

20520454 plus 1453 1454 1455 1456 CYFIP1 ROI 19

42926511 plus 1457 1458 1459 1460 RET ROI 7

14936119 minus 1461 1462 1463 1464 SLC1A6 ROI 4

20498473 minus 1465 1466 1467 1468 CYFIP1 ROI 10

112205714 minus 1469 1470 1471 1472 APC ROI 15

32552476 minus 1473 1474 1475 1476 FHOD3 ROI 16

14940264 minus 1477 1478 1479 1480 SLC1A6 ROI 3

20554187 plus 1481 1482 1483 1484 CYFIP1 ROI 28

14943631 minus 1485 1486 1487 1488 SLC1A6 ROI 2

98393449 minus 1489 1490 1491 1492 TRRAP ROI 38

11115766 minus 1493 1494 1495 1496 FRAP1 ROI 35 179340824 minus 1497 1498 1499 1500 TTN ROI 33

107335174 plus 1501 1502 1503 1504 MGC16169 ROI 18

113315623 minus 1505 1506 1507 1508 CSMD3 ROI 69

51168953 plus 1509 1510 1511 1512 TCF4 ROI 6

197972874 plus 1513 1514 1515 1516 SF3B1 ROI 17

6104065 minus 1517 1518 1519 1520 CHD5 ROI 29

89342303 minus 1521 1522 1523 1524 EPHA3 ROI 3

41925496 plus 1525 1526 1527 1528 ATP5A1 ROI 3

1467225 plus 1529 1530 1531 1532 TPO ROI 8

35137269 minus 1533 1534 1535 1536 ERBB2 ROI 23

150591471 minus 1537 1538 1539 1540 FLG2 ROI 2

10333117 minus 1541 1542 1543 1544 TYK2 ROI 8

74608410 plus 1545 1546 1547 1548 TNNI3K ROI 15

102486426 plus 1549 1550 1551 1552 CDC42BPB ROI 24

11127142 plus 1553 1554 1555 1556 FRAP1 ROI 32

76924180 plus 1557 1558 1559 1560 NAV3 ROI 8

3022903 minus 1561 1562 1563 1564 RIPK1 ROI 2

28380595 plus 1565 1566 1567 1568 NF2 ROI 5

11090085 minus 1569 1570 1571 1572 FRAP1 ROI 55

80777822 minus 1573 1574 1575 1576 TTK ROI 5

151467054 plus 1577 1578 1579 1580 MLL3 ROI 56

98388576 plus 1581 1582 1583 1584 TRRAP ROI 35

14943419 plus 1585 1586 1587 1588 SLC1A6 ROI 2

31818814 plus 1589 1590 1591 1592 BRCA2 ROI 11

79954787 minus 1593 1594 1595 1596 CTNNA2 ROI 5

98316560 plus 1597 1598 1599 1600 TRRAP ROI 1

43650694 plus 1601 1602 1603 1604 SMAD2 ROI 2

55368431 plus 1605 1606 1607 1608 PCDH15 ROI 24

28408859 plus 1609 1610 1611 1612 NF2 ROI 16

197973321 minus 1613 1614 1615 1616 SF3B1 ROI 17

11002256 plus 1617 1618 1619 1620 SMARCA4 ROI 24

151477108 minus 1621 1622 1623 1624 MLL3 ROI 51

10381125 plus 1625 1626 1627 1628 MYH2 ROI 10

26583963 minus 1629 1630 1631 1632 NF1 ROI 26

113416808 minus 1633 1634 1635 1636 CSMD3 ROI 46

173573323 minus 1637 1638 1639 1640 TNR ROI 17

11225881 minus 1641 1642 1643 1644 FRAP1 ROI 7

114036082 minus 1645 1646 1647 1648 CSMD3 ROI 9

26711481 plus 1649 1650 1651 1652 NF1 ROI 57

47871703 minus 1653 1654 1655 1656 MSH6 ROI 2

113726546 minus 1657 1658 1659 1660 CSMD3 ROI 21

11236444 minus 1661 1662 1663 1664 FRAP1 ROI 5

24573476 minus 1665 1666 1667 1668 CDH10 ROI 2

76939708 minus 1669 1670 1671 1672 NAV3 ROI 9

27344847 plus 1673 1674 1675 1676 SUZ12 ROI 11

114100420 minus 1677 1678 1679 1680 CSMD3 ROI 7

148175258 minus 1681 1682 1683 1684 EZH2 ROI 1

197969232 minus 1685 1686 1687 1688 SF3B1 ROI 20

107707428 minus 1689 1690 1691 1692 ATM ROI 48

10158382 plus 1693 1694 1695 1696 VHL ROI 1

55236476 minus 1697 1698 1699 1700 EGFR ROI 26

37807513 minus 1701 1702 1703 1704 GPR124 ROI 7

98388926 minus 1705 1706 1707 1708 TRRAP ROI 35

69722583 minus 1709 1710 1711 1712 BAI3 ROI 5

180404835 plus 1713 1714 1715 1716 PIK3CA ROI 5

997079 plus 1717 1718 1719 1720 ABCA7 ROI 9

3049308 plus 1721 1722 1723 1724 RIPK1 ROI 7

26707974 plus 1725 1726 1727 1728 NF1 ROI 53

7880798 plus 1729 1730 1731 1732 MAP2K7 ROI 3

6138197 minus 1733 1734 1735 1736 CHD5 ROI 4 77107745 plus 1737 1738 1739 1740 NAV3 ROI 33

1405674 plus 1741 1742 1743 1744 TPO ROI 2

26586978 minus 1745 1746 1747 1748 NF1 ROI 29

102479831 plus 1749 1750 1751 1752 CDC42BPB ROI 29

112179021 minus 1753 1754 1755 1756 APC ROI 8

11099597 plus 1757 1758 1759 1760 FRAP1 ROI 47

14928434 minus 1761 1762 1763 1764 SLC1A6 ROI 6

114057092 plus 1765 1766 1767 1768 CSMD3 ROI 8

98347703 minus 1769 1770 1771 1772 TRRAP ROI 16

35129416 plus 1773 1774 1775 1776 ERBB2 ROI 14

51088122 minus 1777 1778 1779 1780 TCF4 ROI 10

98336132 plus 1781 1782 1783 1784 TRRAP ROI 10

48843523 plus 1785 1786 1787 1788 DCC ROI 6

107391014 minus 1789 1790 1791 1792 MGC16169 ROI 4

20496531 minus 1793 1794 1795 1796 CYFIP1 ROI 9

29600343 plus 1797 1798 1799 1800 HM13 ROI 5

35004385 minus 1801 1802 1803 1804 CCNE1 ROI 7

41921923 plus 1805 1806 1807 1808 ATP5A1 ROI 5

81437897 minus 1809 1810 1811 1812 CACNA2D1 ROI 27

113416584 plus 1813 1814 1815 1816 CSMD3 ROI 46

41928966 plus 1817 1818 1819 1820 ATP5A1 ROI 2

148160703 minus 1821 1822 1823 1824 EZH2 ROI 3

20923470 minus 1825 1826 1827 1828 ZNF521 ROI 6

41665436 plus 1829 1830 1831 1832 FOXP4 ROI 9

151495042 minus 1833 1834 1835 1836 MLL3 ROI 41

98368670 plus 1837 1838 1839 1840 TRRAP ROI 25

79938497 plus 1841 1842 1843 1844 CTNNA2 ROI 3

48959180 plus 1845 1846 1847 1848 DCC ROI 9

3030568 minus 1849 1850 1851 1852 RIPK1 ROI 5

98370988 plus 1853 1854 1855 1856 TRRAP ROI 26

64247415 plus 1857 1858 1859 1860 ROR1 ROI 2

79824871 plus 1861 1862 1863 1864 CTNNA2 ROI 2

128630340 minus 1865 1866 1867 1868 SMO ROI 2

85856143 minus 1869 1870 1871 1872 DACH2 ROI 6

49873941 minus 1873 1874 1875 1876 CAMKV ROI 4

11239427 plus 1877 1878 1879 1880 FRAP1 ROI 3

81479421 minus 1881 1882 1883 1884 CACNA2D1 ROI 15

113417907 plus 1885 1886 1887 1888 CSMD3 ROI 45

11983731 plus 1889 1890 1891 1892 MAP2K4 ROI 10

69631833 plus 1893 1894 1895 1896 DLG3 ROI 15

70504023 plus 1897 1898 1899 1900 TAF1 ROI 2

98353085 minus 1901 1902 1903 1904 TRRAP ROI 18

180410107 minus 1905 1906 1907 1908 PIK3CA ROI 6

114359847 plus 1909 1910 1911 1912 CSMD3 ROI 4

11112232 plus 1913 1914 1915 1916 FRAP1 ROI 37

113654756 plus 1917 1918 1919 1920 CSMD3 ROI 25

79954560 plus 1921 1922 1923 1924 CTNNA2 ROI 5

14427278 minus 1925 1926 1927 1928 TRIO ROI 19

69710445 minus 1929 1930 1931 1932 BAI3 ROI 4

81449768 plus 1933 1934 1935 1936 CACNA2D1 ROI 24

107628773 minus 1937 1938 1939 1940 ATM ROI 10

140124267 minus 1941 1942 1943 1944 BRAF ROI 12

55368644 minus 1945 1946 1947 1948 PCDH15 ROI 24

197971473 minus 1949 1950 1951 1952 SF3B1 ROI 18

50663900 plus 1953 1954 1955 1956 ACVR1B ROI 5

42920569 minus 1957 1958 1959 1960 RET ROI 4

25271285 plus 1961 1962 1963 1964 KRAS ROI 2

132335313 plus 1965 1966 1967 1968 NEK11 ROI 5

107392376 minus 1969 1970 1971 1972 MGC16169 ROI 3

112191375 plus 1973 1974 1975 1976 APC ROI 12 70005740 minus 1977 1978 1979 1980 BAI3 ROI 18

48959405 minus 1981 1982 1983 1984 DCC ROI 9

179348985 minus 1985 1986 1987 1988 TTN ROI 25

41240871 plus 1989 1990 1991 1992 CTNNB1 ROI 2

151523874 plus 1993 1994 1995 1996 MLL3 ROI 28

46847475 minus 1997 1998 1999 2000 SMAD4 ROI 8

26691499 plus 2001 2002 2003 2004 NF1 ROI 47

31811428 plus 2005 2006 2007 2008 BRCA2 ROI 9

77118234 plus 2009 2010 2011 2012 NAV3 ROI 37

55370660 minus 2013 2014 2015 2016 PCDH15 ROI 23

28362590 plus 2017 2018 2019 2020 NF2 ROI 2

64330130 plus 2021 2022 2023 2024 MEN1 ROI 5

20492205 minus 2025 2026 2027 2028 CYFIP1 ROI 8

11238642 minus 2029 2030 2031 2032 FRAP1 ROI 4

128632130 plus 2033 2034 2035 2036 SMO ROI 3

21059225 minus 2037 2038 2039 2040 ZNF521 ROI 3

93533046 minus 2041 2042 2043 2044 ROR2 ROI 7

47307518 plus 2045 2046 2047 2048 ARAF ROI 2

81451882 minus 2049 2050 2051 2052 CACNA2D1 ROI 22

48843745 minus 2053 2054 2055 2056 DCC ROI 6

61803515 minus 2057 2058 2059 2060 ARFRP1 ROI 5

80804253 plus 2061 2062 2063 2064 TTK ROI 16

80473697 plus 2065 2066 2067 2068 CTNNA2 ROI 7

41252256 minus 2069 2070 2071 2072 CTNNB1 ROI 8

69637323 plus 2073 2074 2075 2076 DLG3 ROI 21

21059954 minus 2077 2078 2079 2080 ZNF521 ROI 3

173602817 plus 2081 2082 2083 2084 TNR ROI 8

113940400 plus 2085 2086 2087 2088 CSMD3 ROI 12

42920256 plus 2089 2090 2091 2092 RET ROI 4

112144236 plus 2093 2094 2095 2096 APC ROI 5

89707440 plus 2097 2098 2099 2100 PTEN ROI 7

41931986 plus 2101 2102 2103 2104 ATP5A1 ROI 1

179346305 minus 2105 2106 2107 2108 TTN ROI 28

107708733 minus 2109 2110 2111 2112 ATM ROI 50

85290131 plus 2113 2114 2115 2116 DACH2 ROI 1

180401622 plus 2117 2118 2119 2120 PIK3CA ROI 3

35455582 plus 2121 2122 2123 2124 SRC ROI 3

32576663 minus 2125 2126 2127 2128 FHOD3 ROI 19

11198675 minus 2129 2130 2131 2132 FRAP1 ROI 18

37818947 minus 2133 2134 2135 2136 GPR124 ROI 19

98412348 plus 2137 2138 2139 2140 TRRAP ROI 50

64330909 plus 2141 2142 2143 2144 MEN1 ROI 4

74677809 minus 2145 2146 2147 2148 TNNI3K ROI 18

148137330 minus 2149 2150 2151 2152 EZH2 ROI 17

55236884 minus 2153 2154 2155 2156 EGFR ROI 27

173560193 minus 2157 2158 2159 2160 TNR ROI 20

130041431 minus 2161 2162 2163 2164 GPR133 ROI 6

70246761 minus 2165 2166 2167 2168 IL2RG ROI 4

23614616 minus 2169 2170 2171 2172 ERN2 ROI 13

49267211 minus 2173 2174 2175 2176 DCC ROI 26

11111110 minus 2177 2178 2179 2180 FRAP1 ROI 39

46847237 plus 2181 2182 2183 2184 SMAD4 ROI 8

86321665 minus 2185 2186 2187 2188 KLHDC4 ROI 6

74591546 minus 2189 2190 2191 2192 TNNI3K ROI 9

179355950 minus 2193 2194 2195 2196 TTN ROI 16

80806482 plus 2197 2198 2199 2200 TTK ROI 18

132366810 plus 2201 2202 2203 2204 NEK11 ROI 9

76886280 plus 2205 2206 2207 2208 NAV3 ROI 5

11095346 plus 2209 2210 2211 2212 FRAP1 ROI 51

1436343 plus 2213 2214 2215 2216 TPO ROI 5 41253051 plus 2217 2218 2219 2220 CTNNB1 ROI 9

1476451 plus 2221 2222 2223 2224 TPO ROI 10

11954366 plus 2225 2226 2227 2228 MAP2K4 ROI 6

113310011 plus 2229 2230 2231 2232 CSMD3 ROI 71

11109504 plus 2233 2234 2235 2236 FRAP1 ROI 42

74605378 plus 2237 2238 2239 2240 TNNI3K ROI 11

70559641 plus 2241 2242 2243 2244 TAF1 ROI 29

32335743 plus 2245 2246 2247 2248 FHOD3 ROI 4

70247537 minus 2249 2250 2251 2252 IL2RG ROI 2

50664129 minus 2253 2254 2255 2256 ACVR1B ROI 5

6088892 plus 2257 2258 2259 2260 CHD5 ROI 37

64378230 plus 2261 2262 2263 2264 ROR1 ROI 6

89539856 plus 2265 2266 2267 2268 EPHA3 ROI 9

89643757 minus 2269 2270 2271 2272 PTEN ROI 2

180410518 plus 2273 2274 2275 2276 PIK3CA ROI 7

50673985 plus 2277 2278 2279 2280 ACVR1B ROI 9

11193308 plus 2281 2282 2283 2284 FRAP1 ROI 22

179340581 plus 2285 2286 2287 2288 TTN ROI 33

74487581 plus 2289 2290 2291 2292 TNNI3K ROI 3

56934290 minus 2293 2294 2295 2296 PRKAA2 ROI 6

197973813 minus 2297 2298 2299 2300 SF3B1 ROI 16

26688932 minus 2301 2302 2303 2304 NF1 ROI 44

1005248 minus 2305 2306 2307 2308 ABCA7 ROI 20

34995286 plus 2309 2310 2311 2312 CCNE1 ROI 2

148400008 minus 2313 2314 2315 2316 ACVR2A ROI 10

49171873 plus 2317 2318 2319 2320 DCC ROI 17

55178342 plus 2321 2322 2323 2324 EGFR ROI 3

98414370 minus 2325 2326 2327 2328 TRRAP ROI 52

20479700 plus 2329 2330 2331 2332 CYFIP1 ROI 3

85290367 minus 2333 2334 2335 2336 DACH2 ROI 1

69586693 plus 2337 2338 2339 2340 DLG3 ROI 5

113881417 plus 2341 2342 2343 2344 CSMD3 ROI 14

106789743 minus 2345 2346 2347 2348 ST6GAL2 ROI 5

19028260 plus 2349 2350 2351 2352 CABLES 1 ROI 4

148137084 plus 2353 2354 2355 2356 EZH2 ROI 18

122364535 minus 2357 2358 2359 2360 GRIA3 ROI 8

11005347 minus 2361 2362 2363 2364 SMARCA4 ROI 26

122426549 minus 2365 2366 2367 2368 GRIA3 ROI 13

55226966 minus 2369 2370 2371 2372 EGFR ROI 22

58774623 plus 2373 2374 2375 2376 OMA1 ROI 2

107373394 plus 2377 2378 2379 2380 MGC16169 ROI 14

76858850 minus 2381 2382 2383 2384 NAV3 ROI 3

112204035 minus 2385 2386 2387 2388 APC ROI 15

179355478 minus 2389 2390 2391 2392 TTN ROI 17

1773454 minus 2393 2394 2395 2396 FGFR3 ROI 4

179364894 plus 2397 2398 2399 2400 TTN ROI 8

37810417 minus 2401 2402 2403 2404 GPR124 ROI 10

80688784 minus 2405 2406 2407 2408 CTNNA2 ROI 16

113328275 plus 2409 2410 2411 2412 CSMD3 ROI 65

49215480 minus 2413 2414 2415 2416 DCC ROI 22

113432430 plus 2417 2418 2419 2420 CSMD3 ROI 41

26709474 plus 2421 2422 2423 2424 NF1 ROI 55

26507173 minus 2425 2426 2427 2428 NF1 ROI 2

70596178 minus 2429 2430 2431 2432 TAF1 ROI 34

14346046 minus 2433 2434 2435 2436 TRIO ROI 6

69638746 minus 2437 2438 2439 2440 DLG3 ROI 22

51079664 minus 2441 2442 2443 2444 TCF4 ROI 11

89239439 plus 2445 2446 2447 2448 EPHA3 ROI 1

49177526 plus 2449 2450 2451 2452 DCC ROI 18

73522858 minus 2453 2454 2455 2456 PDZRN3 ROI 6 12 77103362 plus 2457 2458 2459 2460 NAV3 ROI 30

19 10996015 minus 2461 2462 2463 2464 SMARCA4 ROI 20

10 55391368 plus 2465 2466 2467 2468 PCDH15 ROI 21

7 148142888 plus 2469 2470 2471 2472 EZH2 ROI 13

19 11002470 minus 2473 2474 2475 2476 SMARCA4 ROI 24

7 55236225 plus 2477 2478 2479 2480 EGFR ROI 26

19 1005419 plus 2481 2482 2483 2484 ABCA7 ROI 21

17 35136191 plus 2485 2486 2487 2488 ERBB2 ROI 21

1 74574232 plus 2489 2490 2491 2492 TNNI3K ROI 7

1 74674202 plus 2493 2494 2495 2496 TNNI3K ROI 16

17 26616383 minus 2497 2498 2499 2500 NF1 ROI 36

22 28400675 plus 2501 2502 2503 2504 NF2 ROI 13

4 1776295 minus 2505 2506 2507 2508 FGFR3 ROI 7

5 14560081 plus 2509 2510 2511 2512 TRIO ROI 55

The 5' end and the 3' end of the capture oliogonucleotides were blocked and did not contain phosphate or hydroxyl groups and 10 thymines were substituted with uracils to facilitate fragmentation and purification of the splint oligonucleotides after circularization. All oligonucleotides were synthesized at the Stanford Genome Technology Center (Stanford, CA). In an alternative design we substituted the central 40bp of the capture oligonucleotide with a sequence comprising the Illlumina® sequencer adapter sequence. This has the advantage of creating amplicons ready for sequencing in a single amplification reaction, thus greatly facilitating the workflow. Illumina® adapter sequences are available to anyone using their products; any approximately 35 bases, designed to allow attachment of the DNA to be sequenced to the surface of the flow cells used. Other sequencing systems would use other adapters.

Targeted Genomic Circularization

High quality genomic DNA from flash- frozen tissues was first sonicated for 10 minutes in the Bioruptor to a size of 500-1000 bps. The hybridication reactions contained 0.5 μg dsDNA or 3-4 μg ddDNA and 50 pM of each of the capture oligonucleotides. After a brief denaturation step, the mixture was incubated in the PGR machine using a touchdown protocol ranging from 70-50°C and 30-60 minutes for each step. Then a mixture of the cleavage enzymes (Exol and Taq) and circularization enzyme (Ampligase or Taq ligase) were added to each tube and the reactions were incubated for 1 hour at 37°C followed by a touchup protocol from 50-72°C for 30 minute at each step. Excess oligonucleotides in the reactions were cleaved by uracil excision. After a brief purification using the Spin-20 columns, the captured DNA fragments were amplified using the high-fidelity Phusion polymerase and either the generic primer (e.g. ID 102) [9] or Illumina PE-primers for 38-39 cycles. The PCR products were purified using the Fermentas kit. Sequencing Library Construction

The captured target DNA amplified with the generic PCR primers were ligated to PE- adapters after "A-tailing" and gel purified. They were then amplified for 10-12 cycles using the PE primers and re-purified from agarose gel. For DNA fragments captured with built-in PE primer sites, they were first purified away from the primer-dimers by gel electrophoresis and re-amplified for 5 cycles using the short PE primers. After quantitation by the SYBR based fluorescence assay, the libraries were sequenced on Illumina HiSeq or GAIIx using standard conditions.

Sequencing 10 pM of PCR amplified library and 1.5 pM of circularized DNA were sequenced using the Illumina Genome Analyzer IIx. Circular library obtained from 1 μg of starting material was introduced to the sequencing experiment. After sample dilution using hybridization buffer, 20% of the prepared sample (representing 200 ng of starting material) was hybridized in the flow cell. Data Analysis

Sequence reads were aligned to the human genome version hgl9 using ELAND software. The target regions were defined as the ranges from each target specific site to 41 bases upstream or downstream of it (depending on the orientation of the capture

oligonucleotide). The interval of 41 bases was selected because the read length in these experiments was 42. In a paired-end experiment the target region contained both ends of the circularized fragments, while single-read sequencing targeted only 3 ' ends of the circularized fragments. To assess the specificity of the capture, the numbers of sequence reads mapping inside and outside the target region were compared. To illustrate the uniformity of the assay, the reads that aligned perfectly with the specific capture sequences were counted. Read counts were then sorted and normalized using the median sequence yield value from each experiment. The genomic distance between the target specific sites indicates the circle size. In addition, guanine and cytosine proportions within the target sites were determined. The present capture oligonucleotide contains two target specific sites and each site was analyzed separately. To analyze the annealing properties during circularization-hybridization reaction, target specific sites within a single capture oligonucleotide as high or low G+C were classified. Circle sizes and G+C proportions with the sequence yields for each

oligonucleotide were then plotted. EXAMPLE 2; Assessment of overall capture coverage

In a proof of principle experiment, we used a set of previously described capture oligonucleotides [9]. Because we had determined that amplicon size was an important parameter for this type of selective circularization, we chose a subset of 628 capture oligonucleotides, each targeting a 150-250 base region. The assay targets a total of 123,982 bases. We compared the yield and the reproducibility of targeting reactions using DNA extracted from either fresh frozen tissue or FFPE blocks of three individuals. Both fresh frozen and FFPE samples are derived from normal colon according to the pathology reports.

The resulting capture amplicons from matched genomic DNA samples derived from either flash- frozen or FFPE material were concatenated using T4 DNA ligase and mechanically fragmented prior to library preparation. Replicate sequencing was conducted in triplicate to identify sequencing specific errors. The fragmented amplicons ligated to a 4-plex paired-end indexing adapters for two samples from individuals 751 and 761 [13]. The four libraries were combined and sequenced in three separate lanes of an Illlumina GAIIx sequencer. For matched samples from individual 780, paired end sequencing was conducted on both the flash tissue and FFPE derived material in separate full sized lanes. Sequence reads were aligned to the human genome reference. Given the replicate sequencing and matched samples, there were a total of 14 separate sequencing data sets. Each was analyzed separately (Table 1). TABLE 1: Capture yield comparison

Overall, the sequence coverage is very reproducible among the replicates for each individual's samples. As noted in Table 1 the sequence coverage at 10X coverage ranges from 79% to 92% and is 5 to 10% lower for the FFPE derived than for the flash tissue derived samples. The uniformity of capture between the two types of starting material and for all three patient's DNA was compared (Figure 2). Approximately 5-10% fewer regions are captured with a sequence coverage greater than 10X in FFPE relative to flash- frozen tissue.

It was determined that the sensitivity of detection of heterozygote SNVs in the targeted resequencing from FFPE versus flash- frozen derived DNA. As described previously, SNV calling from each dataset was conducted [9]. The results of previous published analysis were advantageously used, demonstrating that the variant calling accuracy improves when relying on calls that can be established from both the forward or reverse strand (e.g. double- stranded) [9]. Of the 83 heterozygotes in high quality genomic DNA from flash- frozen tissue, 71 were also called from the FFPE-derived DNA for individual 751 (85%). Similar sensitivity values for the other two patients (84% and 85% respectively for individuals 761 and 780) were obtained.

EXAMPLE 3; Evaluation of sequencing errors from the archival process

Given that matched samples from normal tissue of the same individual are used, differences between the SNV-calling results between FFPE versus flash- frozen derived DNA is attributable to FFPE- induced damage. Sequencing-related errors were eliminated based on the triplicate resequencing of each sample. As previously published, a straightforward statistical method to identify differences between matched samples which were previously applied to normal tumor pairs [9] was developed. At any given sequence position, the present method imposes that the difference in the second most frequent bases between the two samples exceeds 10% for both forward and reverse strand aligning reads. The 14 datasets were analyzed as seven matched pairs comparing sequence data from matched FFPE versus flash-frozen derived genomic DNA samples. The analysis yielded an average of 10.2 FFPE- specific calls (standard deviation being 4.2) per pair within the 102 Kb target (N=73 total positions for all pairs representing 45 unique positions). This results in one false positive call per every 12 Kb of targeted DNA. The FFPE-specific calls are replicated amongst the datasets that were sequenced in triplicate (patients 751 and 761) indicating that these errors were not attributable to the sequencing chemistry or processing but inherently found in the FFPE-derived DNA. There was no overlap between patients amongst these FFPE specific calls.

The pattern of FFPE-specific substitution errors were examined (Table 2). For substitutions, there are twelve combinations when considering all possibilities. Thirty one changes were transitions and 14 were trans versions. Only 4 categories of substitutions among the 12 different substitutions were observed. This represented 44 out of the 45 observed cases. Nearly all of the observed changes obey the consensus G or C - A or T. The C- T and G->A transitions are compatible with cytosine deamination which is a common FFPE processing artifact [10]. TABLE 2: Substitutions specific to targeted resequencing of the FFPE sample

Non-bolded: Transversions

Bolded: Transitions

Consensus: G or C -> A or T

The above table shows that the chemical treatment involved in the FFPE process causes far fewer single base changes than are normally observed between individuals in the form of SNPs. Further, these chemical modifications are predictable as most likely being G - > A or C-> T. This means that the present methodology can be useful in an SNP analysis of genomic DNA from an FFPE sample.

It is noted that while just one position per 12 kb of targeted sequence results in an FFPE specific calls that passed a statistical significance cutoff for significance and was found in both the forward and reverse strands of capture sequence. From either FFPE or flash- frozen derived genomic DNA, a number of positions had suggestions of a variant but were typically seen only the forward or the reverse strand. Using the variant calling method which imposes double- stranded representation, these positions were effectively eliminated as false positive calls (Figure 3). EXAMPLE 4; Optimizing capture oligonucleotide parameters

Having obtained promising results from the initial capture oligonucleotides, an improved bioinformatic pipeline for in silico capture oligonucleotide design was developed. The present design process optimizes the placement of the targeting arms according to the following considerations: (1) it attempts to place the 20 bp targeting arms in positions unique over the genome and that have no single mismatch neighbor, (2) identifying capture arms with GC content between 30% and 60%, (3) the size distribution of the target genomic regions approximating 220 bases in length. The new design process was applied to the targeting 80 exons from six cancer genes. A total of 288 capture oligonucleotides were synthesized for this six gene capture assay and these pooled oligonucleotides were used on three matched normal and tumors samples from the same individual. One DNA sample was obtained from flash- frozen tumor tissue, one sample was obtained from an FFPE section and a third normal DNA sample was obtained peripheral lymphocytes. Significantly improved performance metrics were noted using these optimized capture parameters. Further optimization of the present process was carried out to show amplicon length obtained at different temperatures with the 628 capture oligonucleotides used. Ranges from 50 deg. to 60 deg. annealing temperatures showed no size bias between an amplicon length of 150-250 bp. Annealing temperature of 50 deg. was shown to yield a higher number of amplified targets. Also, consistent coverage across the amplicon lengths between 150 and 250 bp was shown. It was also shown that the process was tolerant of hairpin structures that can form in ssDNA that is being captured by the present capture probes.

As another novel feature, the sequencing library adapter sequences were incorporated into the universal vector sequence. This enabled a sequencing read library with a single amplification step to be generated, thus significantly reducing the complexity of the workflow used for next generation sequencing instruments such as the Illumina HiSeq,

GAIIx, MiSeq, Life Sciences Solid, Ion Torrent, Pacific Biosciences system and the Roche 454 sequencer among others.

The present compositions may be provided in kit form, comprising a set of capture probes and universal oligonucleotides. Primers and a polymerase for amplification may also be included in the kit. CONCLUSION

The above specific description is meant to exemplify and illustrate the invention and should not be seen as limiting the scope of the invention, which is defined by the literal and equivalent scope of the appended claims. Any patents or publications mentioned in this specification are intended to convey details of methods and materials useful in carrying out certain aspects of the invention which may not be explicitly set out but which would be understood by workers in the field. Such patents or publications are hereby incorporated by reference to the same extent as if each was specifically and individually incorporated by reference and contained herein, as needed for the purpose of describing and enabling the method or material referred to.

REFERENCES

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2. Hodges E, Xuan Z, Balija V, Kramer M, Molla MN, Smith SW, Middle CM, Rodesch MJ, Albert TJ, Hannon GJ, McCombie WR: Genome-wide in situ exon capture for selective resequencing. Nat Genet 2007, 39:1522-1527.

3. Okou DT, Steinberg KM, Middle C, Cutler DJ, Albert TJ, Zwick ME: Microarray- based genomic selection for high-throughput resequencing. Nat Methods 2007, 4:907-

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4. Gnirke A, Melnikov A, Maguire J, Rogov P, Leproust E, Brockman W, Fennell T, Giannoukos G, Fisher S, Russ C, et al: Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol 2009. 5. Varley KE, Mitra RD: Nested Patch PCR enables highly multiplexed mutation

discovery in candidate genes. Genome Res 2008, 18:1844-1850.

6. Tewhey R, Warner JB, Nakano M, Libby B, Medkova M, David PH, Kotsopoulos SK, Samuels ML, Hutchison JB, Larson JW, et al: Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 2009, 27:1025-1031. 7. Porreca GJ, Zhang K, Li JB, Xie B, Austin D, Vassallo SL, LeProust EM, Peck BJ, Emig CJ, Dahl F, et al: Multiplex amplification of large sets of human exons. Nat Methods 2007, 4:931-936.

8. Turner EH, Lee C, Ng SB, Nickerson DA, Shendure J: Massively parallel exon

capture and library- free resequencing across 16 genomes. Nat Methods 2009, 6:315- 316. Natsoulis G, Bell JM, Xu H, Buenrostro JD, Ordonez H, Grimes S, Newburger D,

Jensen M, Zahn JM, Zhang N, Ji HP: A flexible approach for highly multiplexed candidate gene targeted resequencing. PLOS one 2011, 6:e21088.

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Korn JM, Kuruvilla FG, McCarroU SA, Wysoker A, Nemesh J, Cawley S, Hubbell E, Veitch J, Collins PJ, Darvishi K, et al: Integrated genotype calling and association analysis of SNPs, common copy number polymorphisms and rare CNVs. Nat Genet 2008, 40:1253-1260.

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Claims

CLAIMS What is claimed is:

1. A composition useful for preparing a population of double stranded DNA molecules from a sample containing single stranded polynucleic acids, comprising:

(a) a plurality of polynucleotide capture probes, wherein individual capture probes each contain (i) capture arms at a 3' end a 5' end of the probe for hybridizing to specific portions of a single stranded polynucleic acid in the sample and (ii) an invariant sequence between the capture arms, whereby a circular structure comprising a specific capture probe and a polynucleic acid having regions complementary to the capture arms is formed;

(b) a plurality of second polynucleotides having a sequence complementary to the invariant sequence;

(c) a 5' exonuc lease;

(d) a 3' exonuclease; and

(e) a ligase.

2. The composition of claim 1 further comprising at least one of (a) PCR amplification and (b) a DNA polymerase.

3. The composition of claim 1, further comprising a sample comprising single stranded polynucleic acids which are fragments of human genomic DNA.

4. The composition of claim 1, further comprising a sample comprising single stranded polynucleic acids which are fragments of human genomic DNA that have been fixed by crosslinking and embedded in a wax.

5. The composition of claim 1, wherein the polynucleotide capture probes in the

composition comprise at least 500 different capture arm sequences.

6. The composition of claim 1, wherein the 5' exonuclease is Exonuclease I.

7. The composition of claim 1, wherein the 3' exonuclease is also a DNA polymerase.

8. The composition of claim 1, wherein the 3' exonuclease is a thermostable DNA

polymerase.

9. The composition of claim 1, wherein the ligase is a thermostable DNA ligase and the circular structure is formed by DNA molecules.

10. The composition of claim 1, wherein the second polynucleotide comprises PCR

amplification sites and the composition comprises PCR primers complementary thereto.

11. The composition of claim 10, wherein the PGR amplification sites are spaced on the second polynucleotides about 120 to 250 bases apart.

12. A method for analyzing single stranded polynucleotides in a sample, comprising the steps of:

(a) adding to the sample a plurality of polynucleotide capture probes, each capture probe containing capture arms complementary to specific portions of a polynucleic acid in the sample and an invariant sequence between the arms, whereby a circular structure comprising a specific capture probe and a polynucleic acid sample molecule is formed;

(b) adding to the sample a plurality of second polynucleotides having a sequence complementary to the invariant sequence and having amplification sites for amplification of a polynucleic acid in a circular structure; and

(c) adding to the sample containing capture probes and second polynucleotides a mixture of a 5' exonuclease, a 3' exonuclease, and a ligase under conditions whereby exonucleases remove bases from the single stranded polynucleotides to form a new 5' end thereof and a new 3' end thereof, and the ligase ligates the new 5' end to the new 3' end.

13. The method of claim 12 further comprising the step of composition of claim 1 further comprising at least one of amplification primers and a polymerase.

14. The method of claim 12, wherein the single stranded polynucleic acids are fragments of human genomic DNA.

15. The method of claim 12, wherein the single stranded polynucleic acids are fragments of human genomic DNA that have been fixed by crosslinking and embedded in a wax.

16. The method of claim 12 wherein the capture probes comprise at least 500 different probes.

17. The method of claim 12, wherein the 5' exonuclease is Exonuclease I.

18. The method of claim 12, wherein the 3' exonuclease is also a polymerase.

19. The method of claim 12, wherein the 3' exonuclease is a thermostable polymerase.

20. The method of claim 12, wherein the ligase is a thermostable DNA ligase and the circular structure is formed by DNA molecules.

21. A method for analyzing single stranded polynucleotides from a sample, comprising the steps of: (a) adding to the sample a plurality of polynucleotide capture probes, each capture probe containing capture arms complementary to specific portions of a polynucleic acid in the sample and an invariant sequence between the arms, whereby a circular structure comprising a specific capture probe and a polynucleic acid sample molecule is formed in the buffer;

(b) adding to the sample a plurality of second polynucleotides having a sequence complementary to the invariant sequence and having amplification sites for amplification of a polynucleic acid in a circular structure; and

(c) adding to the sample containing capture probes and second polynucleotides a mixture of a 5' exonuclease, a 3' exonuclease, and a ligase under conditions whereby exonucleases remove bases from the single stranded polynucleotides to form a new 5' end thereof and a new 3' end thereof, and the ligase ligates the new 5' end to the new 3' end;

(d) adding to the sample a polymerase and polymerase primers; and

(e) conducting a polymerase chain reaction using the polymerase primers for amplification of a portion of a single stranded polynucleotide captured by a corresponding capture probe.

22. The method of claim 21 further comprising the step of sequencing amplified

polynucleotides from step (e).

23. The method of claim 21 wherein the polymerase chain reaction utilizes an annealing temperature of between about 45 degrees Celsius and 55 degrees Celsius.

24. The method of claim 21 wherein the analyzing single stranded polynucleotides from a sample comprises analyzing polynucleotides from a preserved tissue sample or analyzing polynucleotides from a preserved tissue sample and analyzing

polynucleotides from a fresh sample from the same individual.

25. A kit for preparing a composition according to claim 1 comprising:

(a) a plurality of capture probes, each capture probe containing (i) 5' and 3' end capture arms complementary to specific portions of a polynucleic acid in the sample and (ii) an invariant sequence between the capture arms, whereby a circular structure comprising a specific capture probe and a polynucleic acid sample molecule having regions complementary to the capture arms is formed in the buffer; (b) a plurality of second polynucleotides having a sequence complementary to the invariant sequence and having amplification sites for amplification of a polynucleic acid in a circular structure; and

(c) a 5' exonuc lease, a 3' exonuclease, and a ligase.

26. The kit according to claim 25, wherein said kit further comprises at least one of

amplification primers and a polymerase.