WO2014127484A1 - Acides nucléiques de contrôle externe pour le traçage d'échantillons - Google Patents

Acides nucléiques de contrôle externe pour le traçage d'échantillons Download PDF

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Publication number
WO2014127484A1
WO2014127484A1 PCT/CA2014/050124 CA2014050124W WO2014127484A1 WO 2014127484 A1 WO2014127484 A1 WO 2014127484A1 CA 2014050124 W CA2014050124 W CA 2014050124W WO 2014127484 A1 WO2014127484 A1 WO 2014127484A1
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Prior art keywords
seq
spike
sequence
nucleic acid
candidate
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PCT/CA2014/050124
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English (en)
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Robert Holt
Richard Moore
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British Columbia Cancer Agency Branch
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B70/00—Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present disclosure relates to methods and sequences for tracking and verification of samples.
  • the present disclosure provides, in part, unique DNA sequences, termed “spike-in control nucleic acid molecules” or “spike-ins.”
  • the disclosure provides a method for preparing a spike-in control nucleic acid molecule, by providing a candidate oligonucleotide including a known sequence of about 15 to about 25 deoxyribonucleotides; and a random sequence of about 50 to about 500 deoxyribonucleotides, where the known sequence is positioned 3' to the random sequence; providing a priming oligonucleotide comprising a sequence complementary to at least a portion of the known sequence of the candidate oligonucleotide; hybridizing the candidate oligonucleotide and the priming oligonucleotide to form a candidate oligonucleotide/priming oligonucleotide duplex; contacting the candidate oligonucleotide/priming oligonucleotide duplex with a deoxyribonucleotide (DNA) polymerase in the presence of a mixture of deoxyribonucleotides under conditions suitable for
  • the known sequence can include from about 18 to about 22 deoxyribonucleotides.
  • the random sequence can include from about 150 to about 180 deoxyribonucleotides.
  • the candidate oligonucleotide can include about 200 deoxy ribonucleotides.
  • the selected filtering criteria further include verification of one or more of the following criteria: sequence quality, presence of the known sequence, absence of duplicate sequences, or presence of a single sequence.
  • the disclosure provides a spike-in control nucleic acid molecule prepared according to a method as described herein.
  • the disclosure provides a spike-in control nucleic acid molecule including a sequence as set forth in SEQ ID NOs: 1 to 2087.
  • the disclosure provides a vector including a spike-in control nucleic acid molecule as described herein.
  • the disclosure provides a host cell comprising a vector as described herein.
  • the disclosure provides a population of nucleic acid molecules comprising a spike-in control nucleic acid molecule as described herein.
  • the disclosure provides a method for tracking a plurality of samples by adding a unique spike-in control nucleic acid molecule according to claim 6 or 7 or the vector of claim 8 to each sample; processing the sample through a plurality of processing steps; performing DNA sequencing on each processed sample to determine the sequences present therein; comparing the determined sequences of each processed sample to the spike-in control nucleic acid molecule sequence wherein a match indicates that the sample has been tracked through the processing step.
  • the sample may be a biological sample.
  • the sample may be a biological sample including DNA.
  • the disclosure provides a method of identifying a sample, by providing a sample including a unique spike-in control nucleic acid molecule according to claim 6 or 7 or the vector of claim 8; performing DNA sequencing of the sample of (a) to determine the sequences present therein; determining the presence or absence of the spike- in control nucleic acid molecule sequence, wherein the presence of the spike-in control nucleic acid molecule sequence a match provides an identification of the sample.
  • the sample may be a biological sample.
  • the sample may be a biological sample including DNA.
  • the disclosure provides an article of manufacture including the spike-in control nucleic acid molecule as described herein.
  • the article of manufacture may be a container and the spike-in control nucleic acid molecule may be dried onto the walls of the container.
  • the disclosure provides a database including the sequences of a spike-in control nucleic acid molecule as described herein.
  • Fig. 1 is a schematic illustration of a spike-in molecule in the process of preparation
  • FIG. 2 is a flow chart illustrating a method according to the present disclosure
  • Fig. 3 is a graph comparing the observed versus expected control sequence abundance due to intentional sample mixing during an intentional contamination control experiment.
  • Fig. 4 is a graph illustrating the results of a real, unintentional cross- contamination that was detected using a method according to the present disclosure.
  • spike-in control nucleic acid molecules “spike-in molecules” or simply “spike-ins.”
  • the present disclosure provides a population of validated spike-ins that are unique and share little homology with any other known sequences. Such spike-ins can therefore be used in any experiment type.
  • the spike-ins can be added to biospecimens or other samples, thus for example permitting tracking, confirmation of sample identity and/or determination of cross contamination levels.
  • the spike-in approach described herein is simple and accurate, requiring little extra cost or processing, other than the preparation and addition of the spike- ins.
  • the data can be generated along with the sequence of the biospecimen or other sample and is not dependent on the target or lack of similarity within the sample. For example, the methods described herein may be effective to determine a sample swap with technical replicates or monozygotic twins.
  • biological specimen or "biospecimen” is meant any sample that contains
  • DNA such as any organ, tissue, cell, or cell extract isolated from a subject.
  • a biospecimen can include, without limitation, cells or tissue (e.g., from a biopsy or autopsy) from bone, brain, breast, colon, muscle, nerve, ovary, prostate, retina, skin, skeletal muscle, intestine, testes, heart, liver, lung, kidney, stomach, pancreas, uterus, adrenal gland, tonsil, spleen, soft tissue, peripheral blood, whole blood, red cell concentrates, platelet
  • a biospecimen may also include, without limitation, products produced in cell culture by normal or transformed cells.
  • a biospecimen may also include, without limitation, any organ, tissue, cell, or cell extract isolated from a non-mammalian subject or microorganism.
  • a biospecimen may also be a cell or cell line created under experimental conditions, that is not directly isolated from a subject.
  • a biospecimen can also be cell-free, artificially derived or synthesised.
  • Other, non-biological, samples can include forensic samples, chemical samples, pharmaceutical samples or any sample that may for example be vulnerable to counterfeiting or theft.
  • a “spike-in control nucleic acid molecule,” “spike-in molecule” or “spike-in” may be a DNA molecule of about 65 to about 525 bases, or any integer therebetween, including a known sequence of about 12 to about 25 bases, or any integer therebetween, and a random sequence of about 50 to about 500 bases, or any integer therebetween, where the known sequence is positioned 3' to the random sequence.
  • the known sequence may be about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 bases or more.
  • the random sequence may be about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450 or 500 bases or more, or any integer therebetween.
  • the known sequence is about 22 bases and the random sequence is about 178 bases.
  • the spike-in control nucleic acid molecule may be about 180 to about 250 bases, such as 180, 190, 200, 210, 220, 230, 240 or 250 bases or any integer therebetween.
  • the length of the spike-ins may be varied in order to provide the desired degree of diversity, while retaining a length that can be prepared conveniently and without substantial cost.
  • the known sequence can be any DNA sequence, as long as it can hybridize to a primer or "priming oligonucleotide" under standard hybridization conditions, so as to enable the formation of a duplex.
  • the random sequence can be any sequence prepared using standard techniques for random generation of sequences, as long as it has low sequence identity to a naturally-occurring nucleic acid sequence, to avoid cross-reaction with known or natural sequences. For example, if the random sequence could hybridize with a sequence present in the biospecimen or other sample, then it would be considered to "cross react" and would not be a suitable spike-in. Additional filtering criteria can be applied to candidate oligonucleotides to generate suitable spike-in molecules.
  • the random sequence portion of a candidate oligonucleotide can be examined for homopolymer stretches (i.e., sequences of identical bases, such as GGGGG or TTTTTT) of, for example, more than 5, 6, 7 or more, and sequences containing such homopolymer stretches can be discarded.
  • homopolymer stretches i.e., sequences of identical bases, such as GGGGG or TTTTTT
  • Standard hybridization conditions include hybridization conditions as set out, for example, in Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold Spring Harbor, N.Y., and updates thereto. It is to be understood that hybridization conditions may vary according to the specific processing stage and the size of the spike-in molecule.
  • hybridization conditions include hybridization at about 42°C in 50% formamide, 5XSSC, 5XDenhardt's solution, 0.5% SDS and 100 ug/ml denatured carrier DNA followed by washing two times in 2XSSC and 0.5% SDS at room temperature and two additional times in 0.1XSSC and 0.5% SDS at 42°C.
  • low sequence identity is meant that the random sequence has less than about 20% sequence identity, for example, less than about 20%, 15%, 10%, or 5% sequence identity, to a naturally-occurring nucleic acid sequence. In some embodiments, the random sequence has about 0% to about 20% sequence identity, or any value therebetween, to a naturally-occurring nucleic acid sequence. In some embodiments, the random sequence has 0% sequence identity to a naturally-occurring nucleic acid sequence. In some embodiments, the random sequence does not hybridize to a naturally-occurring nucleic acid sequence under the hybridization conditions incurred during processing of a biospecimen or other sample.
  • the random sequence does not hybridize to a naturally- occurring nucleic acid sequence under hybridization conditions appropriate for the size of the spike-in.
  • a sequence may have low sequence identity if it has less than about 30, 40, or 50 contiguous bases in common with naturally-occurring nucleic acid sequence when measured for example using known alignment programs such as BLAT (Kent, W.J., BLAT— the BLAST-like alignment tool, Genome Research 12(4): 656-664 (2002).
  • Naturally- occurring sequences may be found in, for example, public databases such as the NCBI non- redundant database (RefSeq; www[dot]ncbi[dot]nlm[dot]nih[dot]gov/RefSeq/).
  • the spike-ins can be prepared and used using standard techniques known in the art as, for example described in Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold Spring Harbor, N.Y., and updates thereto.
  • candidate oligonucleotides that include a known sequence of about 15 to about 25 bases, or any integer therebetween, and a random sequence of about 50 to about 500 bases, or any integer therebetween, where the known sequence is positioned 3' to the random sequence, can be synthesized.
  • the candidate oligonucleotide can be contacted with a priming oligonucleotide that has a sequence complementary to at least a portion of the known sequence of the candidate oligonucleotide.
  • the exact degree of complementarity is not critical, as long the priming nucleotide is capable of hybridizing to the known sequence of the candidate oligonucleotide so as to form a duplex.
  • the priming oligonucleotide has 99% or 100% sequence identity to the complement of the known sequence.
  • a suitable DNA polymerase and mixture of deoxyribonucleotides e.g., guanine, thymine, adenine, cytosine or analogues thereof
  • a suitable DNA polymerase and mixture of deoxyribonucleotides can be added to the priming oligonucleotide to synthesize a double stranded candidate molecule in a template-dependent manner.
  • the double stranded candidate molecule can be filled in, if necessary, and cloned into a suitable plasmid vector.
  • the plasmid vector can be pcr4.0, although it is to be understood that any appropriate vector can be used.
  • the double stranded candidate molecule can be introduced into a suitable host cell, such as a microorganism (e.g.,E. coli). Individual colonies can be isolated and plasmid DNA prepared and sequenced. It is to be understood that it is not necessary to sequence the entire double stranded candidate molecule; sequencing of a portion of the double stranded candidate molecule may be sufficient. In some embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the double stranded candidate molecule is sequenced.
  • a suitable host cell such as a microorganism (e.g.,E. coli). Individual colonies can be isolated and plasmid DNA prepared and sequenced. It is to be understood that it is not necessary to sequence the entire double stranded candidate molecule; sequencing of a portion of the double stranded candidate molecule may be sufficient. In some embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the double stranded candidate molecule
  • the DNA sequences obtained from the individual colonies can be subject to one or more of the following filtering criteria: low sequence identity to naturally occurring sequences, lack of long homopolymer stretches, sequence quality, presence of the known sequence, absence of duplicate sequences, and/or presence of a single sequence in a clone.
  • the spike-ins can be detectably labelled at any stage of processing, such as during or after synthesis.
  • the spike-ins can be provided or stored as dried plasmid DNA.
  • the spike-ins can be provided in host cells in, for example, glycerol stocks.
  • Spike-in molecules can be provided as a population of unique and distinct molecules. Such a population of spike-ins is replenishible and expandable. In some embodiments, the population may contain at least about 100 to about 10 7 , or any value therebetween, unique and distinct molecules. In some embodiments, the population may contain at least about 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 10000, 50000, 10 5 , 10 6 , 10 7 , or more, unique and distinct molecules. In some embodiments, the population may contain 100 or more of the sequences set forth in SEQ ID NOs: 1 to 2087. In some embodiments, a single spike-in molecule may be provided in a composition, such as biospecimen or other sample.
  • the present methods are suitable for generating large numbers of unique sequences and can be used in situations involving a large number of parallel processes, such that a unique spike-in can be associated with each biospecimen or other sample undergoing processing.
  • the spike-ins can be used in about 400 or more parallel processes, such as about 400 to about 10,000 or more, or any integer therebetween, such as, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, etc.
  • sets or populations of spike-ins can be used in a rolling manner, i.e., a single set or population can be used in a single set of parallel processes, to for example, reduce cross-contamination.
  • spike-in molecule may be used in any application in which conventional barcodes are used.
  • Each unique spike-in molecule can be added directly to each biospecimen or other sample at the time of receipt, or at any stage of processing, including original field collection of samples.
  • a single spike-in molecule is added to a single biospecimen or other sample undergoing processing, such that the biospecimen or other sample can be tracked through each stage of processing subsequent to the addition of the spike-in.
  • the amount of spike-in to be added to a biospecimen or other sample, or to a container may vary according to the desired sensitivity.
  • the processing involves one or more serial transfers of the biospecimen or other sample to different containers or supports.
  • the identity of the biospecimen or other sample containing the spike-in can be verified at a particular stage in processing.
  • tracking is meant the repetitive identification of a sequence, such as a spike-in sequence, through various stages of processing (such as NGS).
  • by “tracking” is meant verification of a sequence, such as a spike-in sequence, at a particular stage of processing and/or storage.
  • the spike-in molecule can be added in a plasmid vector, which can be a circular molecule or can be a linearized molecule.
  • the spike-in molecule is substantially resistant to incidental degradation while processing, unless the degradation is part of the processing step.
  • the spike-ins can be provided at ambient or room temperature, or as dried DNA. The biospecimens and spike-ins are subsequently inseparable and the spike-in DNA is processed along with the original sample DNA through all sample preparation, sequencing and analysis steps.
  • a spike-in can be detected by PCR and/or sequencing at any stage of the process. Primers targeted to the spike-in sequence or the common vector backbone can be used to monitor any stage of sample processing. Methods as described herein may be used in any sequencing platform.
  • the spike-ins are can be used in high throughput sequencing centers that rely on parallel processing of large numbers of samples, which are prone to cross-contamination.
  • biospecimens or other samples may be subject to parallel processing in large numbers and can be tagged with spike-ins as described herein; Such tagged biospecimens or other samples can be tested and tracked at each stage of subsequent processing. Alternatively, such tagged biospecimens or other samples can be tested, and identity verified, at one or more specific stages of subsequent processing, as desired. In some embodiments, biospecimens or other samples can be tracked and/or verified concurrently.
  • the spike-ins are useful during NGS.
  • NGS techniques include, without limitation, sequencing by sythesis, sequencing by ligation, and sequencing by hybridization.
  • NGS platforms include, without limitation, those from lllumina and Life Technologies, Inc.
  • sequence reads derived from the spike-in are generated along with the standard sequencing run and are used to confirm sample identity and determine cross contamination levels. All reads which fail to align to the reference are re-aligned (for example using BWA or BWA-SW) to a reference containing all potential spike-in sequences, allowing for accurate assignment of reads.
  • the spike-ins can be used to identify cross-contamination and sample swaps through the pipeline, and to closely monitor sample identity during NGS.
  • the spike-ins can be used in a rolling manner to, for example, reduce cross contamination from previous processes.
  • about 400 or more spike-ins can be used in parallel.
  • the spike-ins can be used in whole genome sequencing and direct tissue or blood spike-in.
  • the spike-ins may be used for creating audit trails and trackability for forensic or other critical samples.
  • the spike-ins may be added to foodstuffs, drugs or other consumables for tracking and recall purposes.
  • the spike-in sequences may be recorded in an electronic database, such that the database can be queried for a match between the determined sequence of a processed biospecimen or other sample and an entry in the database to, for example, verify the identity of the biospecimen or other sample.
  • the determined sequence(s) of a biospecimen or other sample may be aligned with a first reference sequence from the database to determine the presence or absence of a match. In the presence of a match, the determined sequence(s) of a biospecimen or other sample may be considered to have been tracked or its identity verified.
  • the determined sequence(s) of a biospecimen or other sample may be re-aligned with a second or subsequent reference sequence from the database, until a match is found.
  • the database of spike-in sequences may be used to ensure that the spike-ins can be used in a rolling manner such that the same sequences are not used in subsequent parallel processes in the same facility, to for example, reduce cross- contamination.
  • the database may be stored on a computer.
  • the storage computer or other computer may be capable of analysing and/or manipulating the database to obtain and verify biospecimen or other sample identity.
  • the spike-ins may be provided in articles of manufacture, such as sample collection or processing containers, including without limitation tubes, vials, syringes, slides, wells, etc., such that a biospecimen or other sample may be tagged with the spike-in at the time of initial collection or receipt of the biospecimen or other sample.
  • articles of manufacture such as sample collection or processing containers, including without limitation tubes, vials, syringes, slides, wells, etc.
  • a biospecimen or other sample may be tagged with the spike-in at the time of initial collection or receipt of the biospecimen or other sample.
  • the spike-ins may be coated on the inside of blood collection tubes.
  • the spike-ins can complement conventional barcode tracking and can be used in addition to conventional barcode tracking to verify sample identity and cross- contamination, for example, upon final analysis of sequence data prior to reporting.
  • oligonucleotide templates with 178 random bases plus a 3' tag of 22 known bases was synthesized (IDT).
  • the oligonucleotides were converted to double stranded DNA by polymerase extension, using Phusion DNA polymerase and a primer complementary to the 3' tag of the known 22mer template oligonucleotide (Fig. 1).

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Abstract

La présente invention concerne en partie des séquences d'ADN uniques, appelées « molécules d'acide nucléique de contrôle externe » ou « spike-in ». Les spike-in peuvent être ajoutés à des échantillons biologiques ou autres échantillons, ce qui permet par exemple le traçage, la confirmation de l'identité de l'échantillon et/ou la détermination de taux de contamination croisée.
PCT/CA2014/050124 2013-02-21 2014-02-21 Acides nucléiques de contrôle externe pour le traçage d'échantillons WO2014127484A1 (fr)

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Cited By (11)

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WO2016001736A1 (fr) * 2014-06-30 2016-01-07 Vela Operations Singapore Pte. Ltd. Compositions pour procédés quantitatifs et/ou semi-quantitatifs de détection de mutations
US20210324467A1 (en) * 2016-03-25 2021-10-21 Karius, Inc. Synthetic nucleic acid spike-ins
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