WO2021203461A1 - Position anchoring bar code system for nanopore sequencing library construction - Google Patents

Abstract

Provided are a position anchoring bar code system for nanopore sequencing library construction, a preparation method and a use of the system. The position anchoring bar code system has higher resolution and higher classification accuracy and can remarkably reduce the identification of false positive rate, improving the overall nanopore sequencing precision and reducing sequencing cost.

Description

Position-anchored barcode system for nanopore sequencing library construction

This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on April 9, 2020, the application number is 202010276679.2, and the invention title is "a position-anchored barcode system for nanopore sequencing library construction", all of which The content is incorporated in this application by reference.

Technical field

The invention relates to the field of gene sequencing, in particular to a position-anchored barcode system for nanopore sequencing database construction.

Background technique

At present, there are many clinically infected patients worldwide and various sources of infection. In China, infectious diseases even account for 49% of the total incidence of all diseases. The current routine clinical diagnosis method is the doctor’s empirical judgment plus microscopy, biochemical analysis, etc. to determine the source of infection of the symptoms, but human factors, detection cycle and detection range limitations can easily cause false detections and missed judgments, especially not conducive to acute Diagnosis and treatment of infection. With the vigorous development of high-throughput sequencing and genomics, metagenomic sequencing technology can quickly and comprehensively identify the composition of microorganisms in samples. It is thriving in the field of infection diagnosis, and it is increasingly widely used in the detection of infectious pathogenic microorganisms. It provides a more accurate diagnostic basis for clinical decision-making and follow-up medication.

The development of Illumina second-generation sequencing is in full swing in China, but there are the following problems when applied to microbial detection: First, the read length of second-generation sequencing is less than a few hundred bp, and there will be high homologous sequences between different species of microorganisms, resulting in The accuracy of metagenomic species analysis is poor, and irrelevant microbial information is fed back in the data report, which causes greater diagnostic interference for doctors; secondly, the identification of deeper disease-causing genes and drug-resistant genes requires assembly and splicing of sequencing sequences Therefore, complex analysis requires higher time and capital costs to make up for the read length defect of the second-generation sequencing data; in addition, the second-generation sequencing-related instruments are expensive, cumbersome to operate, high initial investment, and the entire sequencing time is long, which is difficult. Applicable to the needs of acute infections. The third-generation sequencing technology PacBio has greatly improved the read length of sequencing, and can detect long fragment data of 8-12kb, or even 40-70kb, but its disadvantage is that the library construction process is more complicated. Moreover, like the second-generation sequencing, there is the disadvantage of a long sequencing cycle. After a round of sequencing, it takes tens of hours to complete the data offline. With the subsequent analysis time, it is difficult to meet the rapid identification of pathogenic microorganisms.

Nanopore sequencing technology just makes up for the disadvantages of other sequencing platforms, not only reading long sequence fragments, but also short library building and sequencing time. In addition, the equipment is small and portable, and the data generation and bio-information analysis can be real-time, which perfectly solves the limitation of the sequencing site and the delay of report feedback. Therefore, this technology is very suitable for the analysis and identification of clinical infection microbial pathogens. However, the on-board chip for nanopore sequencing is very expensive and the price is very unfriendly to users. Using barcode information to distinguish multiple samples is a common cost-saving strategy for high-throughput DNA sequencing. For each DNA sample, a unique barcode sequence is introduced during the library building process. After multiple barcode DNA samples are sequenced simultaneously in the same flow cell, the sequencing reads are classified according to the barcode sequence to distinguish different samples on the computer. The expensive chip in the nanopore sequencing technology makes multiple samples on the machine have obvious economic advantages, allowing users to share the fixed cost of a flow cell. A series of kits launched by Oxford Nanopore Company provide 12 different barcodes with a length of 24bp. These barcodes are connected to the two ends of the sample DNA sequence during the library construction process and then sequenced on the computer, so that a chip can obtain 12 different samples at the same time. Sequence information. However, when distinguishing samples based on the barcode sequence later, it was found that the 24bp long barcode that comes with the library building kit is seriously confused. The reason is that the Oxford Nanopore Sequencer will cause reads in the process of converting current signals into bases (ie basecalling). The single-base error rate is as high as 10-15%. Therefore, when classifying reads based on barcode sequence in downstream data analysis, cross-contamination of data between samples due to barcode identification errors will result in false positive identification of microorganisms, which will bring clinical decision-making. It was extremely troubled.

Based on this, the present invention is proposed.

Summary of the invention

The technical problem to be solved by the present invention is to improve the accuracy of the barcode comparison process of the existing nanopore sequencing data sample.

Considering that the barcode comparison of samples on the nanopore sequencing platform often causes errors, which greatly affects the subsequent data processing procedures. The invention categorizes and statistically analyzes the errors that occur during sequence comparison of the nanopore sequencing platform by digging in-depth large amounts of data, and quantifies the influence of different error rate types on sequence identification. Surprisingly, I found that the sequencing error of the indel type (Indel) will greatly increase the error rate of sequence identification, while the base mismatch type (Mismatch) has little effect on the improvement of the error rate of sequence identification, so it is blindly improved in the design of sample barcodes. The length of the barcode has a limited effect on the accuracy improvement, and adding a position anchor sequence to the barcode of the same length improves the accuracy even more. Based on this discovery, the present invention constructs a set of "position-anchored barcode system" containing position-anchored sequences, and validates it based on Nanopore’s library building kit SQK-PBK004, and builds a library of 10 pure bacteria on the computer. The original barcode system and the position-anchored barcode system are used to classify and compare the off-machine data. The results show that the position-anchored barcode system has better sample classification accuracy, which is more than 3 orders of magnitude higher than the original barcode system.

Therefore, the first object of the present invention is to provide a position-anchored barcode system that improves the resolution accuracy of sample nanopore sequencing.

The second object of the present invention is to provide a preparation method and application of the above-mentioned position-anchored barcode system.

In order to achieve the above objectives, the present invention provides the following technical solutions:

The present invention provides a position-anchored barcode system for nanopore sequencing library construction, which is characterized in that the system includes the following structure:

[BARCODE-ANCHOR] _n -BARCODE _n+1

Where n≥1,

The BARCODE is a barcode sequence,

The ANCHOR is an anchor sequence.

In some embodiments, the system includes the following structure: FLANK1-[BARCODE-ANCHOR] _n -BARCODE _n+1 -FLANK2,

The FLANK is a flanking sequence,

In some embodiments, the 1≤n≤10; preferably, the n is 1,2,3.

In some embodiments, the BARCODE sequences are the same or different; preferably, the BARCODE sequences are different;

In some embodiments, the ANCHOR sequences are the same or different; preferably, the ANCHOR sequences are different.

In some embodiments, the length of the ANCHOR sequence is 5-50 bp; preferably, the length of the ANCHOR sequence is 10-35 bp;

In some embodiments, the homology between the ANCHOR sequence and the BARCODE sequence is <70%; preferably <50%.

In some embodiments, the length of the FLANK is 10-30 bp; preferably, the length of the FLANK sequence is 15-25 bp;

In some embodiments, the position-anchored barcode system for nanopore sequencing library construction is characterized in that the system includes any of the following structures:

FLANK1-BARCODE ₁ -ANCHOR ₁ -BARCODE ₂ -FLANK2;

FLANK1-BARCODE ₁ -ANCHOR ₁ -BARCODE ₂ -ANCHOR ₂ -BARCODE ₃ -FLANK2;

FLANK1-BARCODE ₁ -ANCHOR ₁ -BARCODE ₂ -ANCHOR ₂ -BARCODE ₃ -ANCHOR ₃ -BARCODE ₅ -FLANK2;

In some embodiments, the ANCHOR sequences are different or the same, preferably the ANCHOR sequences are different;

In some embodiments, the BARCODE sequences are different or the same, preferably the BARCODE sequences are different.

The present invention also provides a method for preparing the above-mentioned position-anchored barcode system for nanopore sequencing library construction, characterized in that: the method includes directly synthesizing the nucleotide sequence of the position-anchored barcode system, or through analysis After the segments are synthesized, they are connected to prepare the position-anchored barcode system.

In some embodiments of the present invention, when preparing a position-anchored barcode system containing existing barcode adapters, the preparation method is as follows: on the basis of the existing nanopore sequencing library barcode, bridge primers are used to realize the existing barcode adapter and The series of barcode linkers is designed; preferably, the bridging primer sequence is ANCHOR in the structure of the position-anchored barcode system.

In some embodiments of the present invention, the existing barcode connector is derived from the original barcode of the SQK-PBK004 kit of ONT.

The present invention also provides an application of the above-mentioned position-anchored barcode system for nanopore sequencing library building in improving the accuracy of sequencing samples classification.

The present invention also provides an application of the above-mentioned position-anchored barcode system for nanopore sequencing library building in reducing false positives of sequencing samples.

The invention also provides an application of the above-mentioned position-anchored barcode system for nanopore sequencing library construction in sequencing library construction.

The present invention also provides an application of the above-mentioned position-anchored barcode system for nanopore sequencing library construction in sequencing.

The present invention also provides a method for constructing a sequencing library, which is characterized in that the position-anchored barcode system of the above-mentioned nanopore sequencing library is used to construct a sequencing library.

The present invention also provides a sequencing adapter, characterized in that the sequence of the sequencing adapter includes the position anchor barcode system described above.

The present invention also provides a composite, characterized in that the composition is connected to the above-mentioned position-anchored barcode system.

The present invention also provides a composition, characterized in that the composition contains the above-mentioned position-anchored barcode system.

The present invention also provides a kit for nanopore sequencing library construction, characterized in that the kit contains the above-mentioned position-anchored barcode system or the above-mentioned sequencing adapter.

The beneficial technical effects of the present invention:

1) The present invention proves for the first time that indel type errors are the main reason for overall sequence alignment errors. In contrast, base mismatch types have less impact on overall sequence alignment errors. In practice, the present invention restricts indel type errors from extending to the overall comparison result by introducing an anchor sequence in the barcode system, greatly reduces the degradation of the comparison score caused by indels, and screens out long-distance barcode interference to achieve Accurate bar code resolution; compared to only increasing the length of the bar code sequence, although increasing the bar code length can appropriately reduce sample classification errors caused by base mismatches, the accuracy of the overall sequence comparison result is very limited. The position-anchored barcode system has an extremely significant effect on improving the accuracy of the results.

2) The present invention is based on the library building process of the nanopore platform SQK-PBK004, cleverly uses its built-in barcode to connect the independently developed barcode sequence, and uses the connecting part sequence as the anchor sequence to design FLANK1-BARCODE ₁ -ANCHOR ₂ -BARCODE _2- FLANK2 type position anchoring barcode system, this system can improve the classification accuracy from 0.999 to 0.999999 when distinguishing different samples.

3) In practical applications, the position-anchored barcode system of the present invention can design barcodes of different lengths and the number of anchor sequences according to different requirements, so as to achieve the balance of classification accuracy and microbial detection rate for different requirements.

4) The position-anchored barcode system of the present invention has better resolution, higher accuracy, and reduced false positive identification, can improve the accuracy of nanopore sequencing as a whole, reduce sequencing costs, and is suitable for popularization and use.

Description of the drawings

In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the specific embodiments or the description of the prior art. Obviously, the appendix in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work.

Figure 1. Error rate statistics based on the sequencing data of the kit barcode system; Figure A shows the average error rate and median error rate of each site in the actual sequencing of the barcode adapter sequence of the 10 sets of kits; Figure B shows the barcode of the 10 sets of kits In the actual sequencing of the adapter sequence, the average error rate and the median error rate of the three types of errors of insertion, deletion, and mismatch at each site are aligned; Figure C shows the corresponding relationship between the barcode adapter site of the kit and the alignment error; Figure D shows A summary of the subcategories of the alignment error types of the barcode adapter in the kit, which shows the distribution of alignment errors that occur at different sites. The abscissa is the base position of the barcode sequence, and the ordinate is the error type. The color of the small cells in the figure indicates that The error rate of the error type at the site, the darker the color means the higher the error rate. The color blocks in the Block comment area indicate different components of the connector sequence, and the error type is clustered by Euclidean distance;

Figure 2. The influence of different alignment error types on the accuracy of overall sequence classification; Figure A shows the total error rate is 8%; Figure B shows the total error rate is 16%;

Figure 3. The influence of no anchor sequence (ANCHOR=0 group), 1 anchor sequence (ANCHOR=1 group), and 2 anchor sequences (ANCHOR=2 group) on the overall accuracy of barcode alignment;

Figure 4. Schematic diagram of the original and optimized database construction process;

Figure 5. Comparison of sample classification accuracy between the original barcode system and the position-anchored barcode system. The shaded part indicates the result of accurate classification, and the other results are the result of misclassification.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

Definition of some terms

Unless otherwise defined below, the meanings of all technical and scientific terms used in the specific embodiments of the present invention are intended to be the same as those commonly understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

As used in the present invention, the terms "including", "including", "having", "containing" or "involving" are inclusive or open-ended, and do not exclude other unlisted elements or method steps . The term "consisting of" is considered a preferred embodiment of the term "comprising". If in the following a certain group is defined as comprising at least a certain number of embodiments, this should also be understood as revealing a group preferably consisting of only these embodiments.

The terms "approximately" and "generally" in the present invention represent the accuracy range that can be understood by those skilled in the art and can still guarantee the technical effect of discussing the feature. The term usually indicates a deviation of ±10% from the indicated value, preferably ±5%.

The indefinite or definite article used when referring to a noun in the singular form such as "a" or "an", "the" includes the plural form of the noun.

In addition, the terms first, second, third, (a), (b), (c) and the like in the specification and claims are used to distinguish similar elements, and are not necessary for the order of description or time. It should be understood that the terms so applied are interchangeable under appropriate circumstances, and the embodiments described in the present invention can be implemented in other orders than described or exemplified in the present invention.

The following terms or definitions are only provided to help understand the present invention. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

Some technical terms in the present invention are explained as follows:

The "position-anchored barcode system" of the present invention refers to a multi-barcode sequencing tag system containing two or more BARCODE sequences in series. The BARCODEs are anchored by a specific ANCHOR sequence. This system can be applied to nanopore sequencing. The construction of sequencing library in, can improve the accuracy of sequencing samples classification, and reduce the application of sequencing sample classification false positives. Its specific structure can be the [BARCODE-ANCHOR] _n -BARCODE _n+1 described in the present invention, where n≥1, the BARCODE is a barcode sequence, and the ANCHOR is an anchor sequence. It can be understood that any composition, compound, or system including the above structure is within the scope of the present invention. Although the present invention is explained by taking the barcode of the SQK-PBK004 kit in the prior art as an example, it is only an exemplary description and does not limit the present invention. The present invention has passed specific bio-information theory analysis and wet experiment verification, which proves that any _{barcode system containing [BARCODE-ANCHOR] n} -BARCODE _n+1 structure can be used for the construction of sequencing library, which can improve the accuracy of sequencing samples classification , To reduce the false positives of sequencing samples. In some preferred embodiments of the present invention, the structure of the position-anchored barcode system may be as follows: FLANK1-[BARCODE-ANCHOR] _n -BARCODE _n+1 -FLANK2, the FLANK is a linker sequence, and the linker sequence is a sequencing library The construction of conventional components can be understood in this field. FLANK1 and 2 can be the same or different in sequence according to actual needs.

In view of the fact that by digging a large amount of data in-depth in the embodiments of the present invention, the errors in the sequence comparison of the nanopore sequencing platform are classified and statistically analyzed, and the influence of different error rate types on sequence identification is quantified, and the indel type (Indel ) Sequencing errors will greatly increase the error rate of sequence identification, and the type of base mismatch (Mismatch) has little effect on the improvement of the error rate of sequence identification. Therefore, in the design of sample barcodes, blindly increasing the length of the barcode will improve the accuracy. The impact is limited. In addition, some examples of the present invention have also confirmed the problem of sequence length. For example, in Example 2, it is mentioned that "in order to achieve an overall alignment accuracy of 99.99%, when a base mismatch type error of 0.16 is introduced, the barcode length only needs to reach 40bp; when the indel type error of 0.16 is introduced, the barcode length needs to reach 80bp". It can be seen that the length of the position-anchored barcode system of the present invention is appropriately selected in the field according to actual needs. For example, in some embodiments of the present invention, the 1≤n≤10, such as n=1, 2, 3, 4, 5. ,6,7,8,9,10; Preferably, the n is 1, 2 or 3.

It is understandable that BARCODE is used as a marker sequence in sequencing. In the position-anchored barcode system of the present invention, the sequence can be the same or different; in some preferred embodiments, the BARCODE sequence is different. . Similarly, the ANCHOR sequence is used as an anchor component, and the starting sequence may be the same or different. In some preferred embodiments, the ANCHOR sequence is different. In addition, the length of the ANCHOR sequence can be a length known in the art, for example, it can be 5-50 bp. In some preferred embodiments, the length of the ANCHOR sequence is 10-35 bp.

The ANCHOR sequence is used as the anchor component of BARCODE. Its sequence should be distinguished from the BARCODE sequence. There is no special restriction. The homology between the ANCHOR sequence and the BARCODE sequence can be <80%, <70%, <60%, <50%, < 40%, <30%, <20%, <10%; in some preferred embodiments, the homology is <50%.

It can be understood that, as some exemplary position-anchored barcode systems of the present invention, the structure can be specifically as follows:

FLANK1-BARCODE ₁ -ANCHOR ₁ -BARCODE ₂ -FLANK2;

FLANK1-BARCODE ₁ -ANCHOR ₁ -BARCODE ₂ -ANCHOR ₂ -BARCODE ₃ -FLANK2;

FLANK1-BARCODE ₁ -ANCHOR ₁ -BARCODE ₂ -ANCHOR ₂ -BARCODE ₃ -ANCHOR ₃ -BARCODE ₄ -FLANK2;

FLANK1-BARCODE ₁ -ANCHOR ₁ -BARCODE ₂ -ANCHOR ₂ -BARCODE ₃ -ANCHOR ₃ -BARCODE ₄ -ANCHOR ₄ -BARCODE ₅ -FLANK2;

......

The "barcode connector" in the present invention refers to a complete section containing a barcode sequence and flanking sequences at both ends. For example, in the embodiment of the present invention, the self-designed barcode connector is defined as a BBRCD connector, and the barcode connector in the original kit SQK-PBK004 is defined as an ABRCD connector.

The "bar code sequence" in the present invention refers to a specific sequence of a bar code, which is included in the bar code connector and is a part of the sequence of the bar code connector. For example, in the embodiment of the present invention, the independently designed barcode sequence is defined as BBRCD, and the barcode sequence in the original kit SQK-PBK004 is defined as ABRCD.

The "anchor sequence (ANCHOR)" in the present invention refers to a nucleotide sequence used to anchor BARCODE. Its length can be any verified length known in the art, for example, it can be 5-50 bp. In the embodiment, it can be 10-35bp; its sequence should be distinguished from the BARCODE sequence, and there is no particular limitation. The homology of ANCHOR sequence and BARCODE sequence can be <80%, <70%, <60%, <50%, <40 %, <30%, <20%, <10%; in some preferred embodiments, the homology is <50%. Exemplarily, for example, the ANCHOR sequence mentioned in the embodiment of the present invention includes SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 13, and the like.

The "FLANK" in the present invention refers to the flanking sequences at both ends of the barcode system, which are conventional components of sequencing barcode adapters. For example, for a nanopore sequencing platform, FLANK1 in the present invention is a Y-type sequencing adapter that connects the motor protein. Ensure that the DNA passes through the nanopore to achieve normal sequencing; FLANK2 is used to connect the sequence of the sequencing sample, and its length can be any verified length known in the art, for example, it can be 10-30 bp, and in some preferred embodiments, it can be 15-25 bp . Exemplarily, such as the FLANK sequence SEQ ID NO. 16 and SEQ ID NO. 26 mentioned in the embodiment of the present invention.

The present invention is further described by the accompanying drawings and the following examples. The accompanying drawings and examples are only to illustrate specific embodiments of the present invention and should not be construed as limiting the scope of the present invention in any way. Unless otherwise specified, the experimental methods disclosed in the present invention all adopt conventional techniques in this technical field, and the reagents and raw materials used in the examples are all commercially available.

Example 1 Comparison error statistics

The present invention considers that the main reason for the confusion of the barcode is the sequence difference between the sequenced barcode and the preset real barcode, so the difference between the barcode sequence obtained by the sequencing and the real barcode sequence is sorted and sorted first. To this end, the present invention uses 10 sets of barcodes of ONT's SQK-PBK004 kit to build a separate library of sample DNA, intercept 250bp of the 5'end of the sequencing data to ensure that the barcode region is included, and then perform a global connection with the corresponding preset barcode adapter. Overlap alignment. Finally, according to the output multiple alignment file, the comparison difference of each position of the barcode adapter sequence is summarized, and the error position distribution and error type of the sequencing barcode are counted. Among them, the error types are divided into three categories, namely insertion (I), deletion (deletion, D) and base mismatch (mismatch, X).

Results: First of all, overall, after the 10 sets of sequencing data were initially filtered in the fastq format, a total of 285,43061 reads, but the actual number of reads participating in error statistics was only 24,075,634, so about 15.65% of the results were filtered out in the error statistics process. Reads. In the actual sequencing of the 10 groups of barcode sequences participating in the statistics, the average error rate of each site was 8.01%, and the median error rate was 5.80% (Figure 1A). The average error rate of one group of comparison data was as high as 10.31%, and the average error The group with the lowest rate also has 6.53%.

Secondly, starting from the three types of alignment errors (Figure 1B), the average insertion error rate in the actual sequencing of 10 sets of barcode sequences is 2.28%, the average deletion error rate is 3.58%, and the average base mismatch The error rate was 2.16%; the median error rates were 1.70%, 2.16% and 1.57%.

From Fig. 1B, there is a significant difference between the average and median error rates of each point of the barcode connector, so the present invention further summarizes the alignment errors of different error types at different barcode sequence positions. Figure 1C shows that, except for the high error rate of the first 6 bases at the 5'end caused by the initial instability of sequencing, there is no significant error rate difference in the remaining positions.

Furthermore, since the position has little influence on the error type, the present invention carefully summarizes the error type subclasses without considering the position (Figure 1D): the preset base of the reference sequence plus the mutation of the query sequence Bases such as GA indicate that G is mismatched as A; deletions in alignment sites are indicated by missing bases and subtitles D, such as GD; insertions in alignment sites are indicated by I, and the inserted bases are appended to I, such as IC The insertion of base C occurs at the site; 2 insertions are I2, and 3 or more insertions are II; after the alignment mismatch occurs at the site, another base insertion occurs, which is indicated by X2, and 2 or more insertions occur. For XX. According to the distance of different error types in Figure 1D, except for the GA and AG mismatch types, the mismatch probability between other bases is similar, and there is no obvious bias; observing the 4 lines with the deepest average chromaticity, it is found that there are exactly 4 bases The average error rate for the comparison of the missing probability is about 0.09, and there is no obvious base missing bias; finally, the error type of the comparison insertion is observed. The 5 rows with the most uniform color block distribution indicate that the probability of inserting different single bases is close. And it is at the same cluster level as the error type I2 of inserting 2 bases. In summary, the data for different error types subcategories are as follows:

Table 1.

Type of error	Error rate
D	0.03580
I1	0.01575
XN	0.01340
GA	0.00548
AG	0.00267
I2	0.00368
II	0.00130
X2	0.00172
XX	0.00032

In the above table, XN represents all mismatch types except GA and AG mismatches, I1 represents a single base randomly inserted, I2 and II represent randomly inserted 2, 3 to 5 bases, and 3, 4 respectively. , The ratio of 5 bases is 8:1.8:0.2; X2,XX indicates that the site randomly introduces unmatched bases and then inserts one or more bases.

Example 2 The influence of different error types and barcode length on the overall accuracy of barcode comparison

According to the different error types and corresponding error rate values of the above-mentioned embodiment 1, the present invention simulates a total of 6 groups of barcode sequences with lengths of 20bp, 40bp, 60bp, 80bp, 100bp, and 120bp, each of which simulates 12 different barcode elements. . The present invention takes 80bp as an example to illustrate the specific overview of the simulation. First, the present invention presets 12 ideal barcode sequences, and the sequence information is shown in Table 2:

Table 2.

Then three different types of errors are introduced with the probability of a total error rate of 0.08 for each site, which are indels only, base mismatches, and both indels and base mismatches. The detailed error rate distribution ratio of each error type is consistent with Table 1, and the total error rate is increased or decreased year-on-year. A flow cell has multiple samples on the machine, there will be multiple barcode DNA in the actual off-machine data, and the barcodes between the samples will be confused with each other. The present invention simulates 100,000 joint sequences for each preset barcode under each set of length, and all simulated The sequence is mixed together to simulate the situation that 12 samples are on the machine at the same time. The analog sequence name uses the preset barcode name itself plus a digital number. The data is classified through the same biometric analysis process, and finally the barcode information obtained by the classification is compared with the analog sequence name. Determine whether crosstalk occurs.

Result: As shown in Figure 2A, from Figure 2A, when the total error rate is 0.08, regardless of the type of error introduced, as the length of the barcode increases, the accuracy of the overall alignment of the barcode sequence gradually increases; for the same length, The accuracy rate of the overall alignment of the barcode sequence that introduces the wrong type of indel is significantly lower than that of the overall alignment of the barcode sequence that introduces the wrong type of base mismatch. These two conclusions are still valid when the overall error rate is increased to 0.16 (Figure 2B). For example, in order to achieve an overall alignment accuracy rate of 99.99%, when a base mismatch type error of 0.16 is introduced, the bar code length only needs to reach 40 bp; while an indel type error of 0.16 is introduced, the bar code length needs to reach 80 bp. It can be seen that the comparison of indel type errors has a greater impact on the accuracy rate than base mismatches, while the comparison of barcode length has a limited impact on the accuracy rate.

Example 3 The influence of inserting anchor sequence on the overall accuracy of barcode comparison

Based on the conclusion of the above embodiment 2, the present invention intends to modify the barcode fragment in the following two ways, taking 80bp as an example:

1) Insert an anchor sequence: replace the 12 bases in the middle of the barcode sequence with the anchor sequence with the same 12bp sequence (that is, underlined in Table 2), that is, replace the original preset barcode fragment with the same length "short barcode- Anchor sequence-short barcode" form;

2) Insert two anchor sequences: replace the 20bp at both ends of the barcode sequence in Table 2 with 12bp anchor sequence 1 and anchor sequence 2 (shown in bold in Table 2), that is, short barcodes of the same length- Anchor Sequence 1-Short Barcode-Anchor Sequence 2-Short Barcode.

The position range of the short bar code is locked by the position information obtained in the comparison process of the anchor sequence, and finally the data is classified according to the result of the short bar code combination.

Result: Replace the underlined 12bp in Table 2 with the specific ANCHOR anchor sequence GGTGCTGTTAAC (SEQ ID NO.50), introduce the error type and error distribution in Example 1 at each location, and the total error rate is randomly distributed Within the range of E±0.5E (where the value of E is 0.08, 0.12, 0.16), each E value simulates 60 million bar code sequences;

In the same way, the 12bp of the bolded sequence in Table 2 is replaced with the anchor sequence GGTGCTGTTAAC (SEQ ID NO.50) and the anchor sequence GTACGGAAGTCG (SEQ ID NO.51) for sequence simulation.

Subsequently, in the comparison process, the data is classified by using the anchor sequence to lock the barcode position range, and the non-anchor sequence (ANCHOR=0 group), 1 anchor sequence (ANCHOR=1 group) and 2 anchor sequences are used. The classification of (ANCHOR=2 groups) is accurately determined for comparison. It can be seen from Figure 3 that after considering the anchoring of the sequence, the classification accuracy is significantly improved:

When the E value is 0.08, the classification accuracy rate of the 80bp ANCHOR=0 group can only reach 99.9999%; the classification accuracy of the ANCHOR=1 group and the ANCHOR=2 group containing the anchor sequence are both improved to 100%;

When the E value is increased to 0.12, the resolution level is sorted as follows: ANCHOR=2 group>ANCHOR=1 group>ANCHOR=0 group;

When the E value is increased to 0.16, the difference in classification accuracy of the three increases, and for the simulation data generated by the present invention, the classification accuracy of the ANCHOR=2 group is still 100%.

According to Examples 2 and 3, increasing the barcode length can improve the sample resolution accuracy to a certain extent, but the improvement rate gradually decreases with the increase of length, and it is difficult to achieve an order of magnitude increase; by inserting an anchor sequence to anchor the barcode area, the ratio can be significantly reduced. The indels in the alignment are reduced, and the simulation data can reach 100% accuracy. With the increase of anchor sequences, the accuracy of data classification also increases significantly. According to the 10-15% error rate of a single base during the basecall process of the Oxford Nanopore Sequencer and the total number of reads for a complete sequence, it is speculated that the barcode sequence in the actual offline sequencing data can be at least 3 orders of magnitude correct by introducing an anchor sequence The rate increases.

Example 4 Preparation of Position Anchor Barcode System and Library Construction

In order to further verify the above theory through experiments, this embodiment is taken as an example, based on the original ONT company’s nanopore library PCR barcode kit SQK-PBK004, clever use of its existing single barcode adapter (sequence structure FLANK1-ABRCD -FLANK3'), through the connection reaction, connect it with the barcode adapter (sequence structure FLANK5'-BBRCD-FLANK2) independently designed in the present invention to form the position-anchored barcode system of the present invention: FLANK1-ABRCD-ANCHOR- BBRCD-FLANK2 (where the ANCHOR sequence is the sequence after FLANK3' and FLANK5' are connected by a ligation reaction). FLANK2 continues to connect with the sample DNA of known source through the ligation reaction, so that the final DNA to be sequenced has a position-anchored barcode system. This design allows us to infer the connected barcode sequence based on the results of the known sample DNA, thereby quantifying the classification accuracy of the barcode system.

The specific construction process is as follows:

According to the barcode sequence information of other kits of ONT company, the present invention obtains 10 barcode sequences BBRCD with excellent distinguishability from each other through combinatorial comparison; and then adds a 13bp conservative flanking sequence FLANK to the 5'end. The sequence has good PCR primer characteristics, moderate GC content, no hairpin and dimer structure, etc., and the Y-shaped structure of the original PCR linker is used to avoid the situation where multiple barcodes are connected in series in the connection step; ANCHOR sequence, in this experiment It is also the 3'end sequence (SEQ ID NO.13) of the PCR bypass primer, which is consistent with the self-designed barcode adapter FLANK5', and the 5'end base sequence is consistent with the FLANK3' of the original barcode adapter of the kit, thus achieving a The PCR reaction obtains a sequenced DNA fragment with 5'and 3'ends in series with double barcode adapters simultaneously.

The PCR bridging primer sequence is as follows:

F1: 5'-TTCTGTTGGTGCTGATATTGC CCGACTTCCGTAC -3' (SEQ ID NO.13)

F2: 5'-ACTTGCCTGTCGCTCTATCTTC CCGACTTCCGTAC -3' (SEQ ID NO.14)

Note: The underline indicates the 13bp sequence consistent with the self-designed barcode linker FLANK5'.

The sequence of the self-designed barcode connector FLANK5'-BBRCD-FLANK2 is shown in Table 3:

table 3

The sequence of the original barcode adapter FLANK1-ABRCD-FLANK3' of the SQK-PBK004 kit is shown in Table 4:

Table 4

This example is aimed at 10 cases of standard pure bacteria strains Brevibacillus borstelensis, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica, Klebsiella pneumoniae, Listeria monocytogenes, Staphylococcus aureus; Acinetobacter baumannii, Stemallophila subtilis, and the library optimization diagrams used in the construction process Each pure bacteria sample introduces a different position-anchored barcode sequence for library preparation, and specifically prepares 10 sets of position-anchored barcode sequences, as shown in Table 5.

table 5

Location anchoring

ABRCD

BBRCD

Barcode sequence	To	To
barcode01	ABRCD01	BBRCD01
barcode02	ABRCD02	BBRCD02
barcode03	ABRCD03	BBRCD03
barcode04	ABRCD04	BBRCD04
barcode05	ABRCD05	BBRCD05
barcode06	ABRCD06	BBRCD06
barcode07	ABRCD07	BBRCD07
barcode08	ABRCD08	BBRCD08
barcode09	ABRCD09	BBRCD09
barcode10	ABRCD10	BBRCD10

The specific nucleotide sequences of the 10 sets of anchor barcodes are shown in Table 6, wherein the underlined part is the ANCHOR sequence.

Table 6

The specific library preparation steps are as follows:

(1) Annealing

1. Dilute the lyophilized powder of the above synthesized linker with annealing solution (1mM EDTA; 50mM NaCl; 5mM Tris-HCl pH 7.5) to 100μM;

2. Mix the complementary strands equimolarly (take 4ul each), incubate at 95°C for 5min, slowly cool down to room temperature (about 25°C) on the PCR machine;

3. Use annealing solution to dilute the annealed joints to 640mM with annealing solution;

4. Store at 4°C for later use.

(Two), end repair

1. Take 100ng of nucleic acid sample from 0.2ml PCR tube and add water to make up to 50μl

2. Add 7μl Ultra II End-prep reaction buffer, 3μl Ultra II End-prep enzyme mix, rotate and mix at room temperature, incubate at 20°C for 5 minutes, incubate at 65°C for 5 minutes, and then leave it at room temperature;

3. Add 1×beads (AMPure XP beads), rotate and mix at room temperature, incubate for 5 min, and then put on the magnetic stand to remove the supernatant;

4. Wash the beads twice with 200μl of freshly prepared 80% alcohol;

5. Add 17μl nuclease-free water, rotate and mix at room temperature and incubate for 2min, and then put it on the magnetic stand until it is clear;

6. Carefully pipette 15μl of supernatant into 0.2ml PCR tube

(Three), joint connection

1. Add 10μl of the diluted adapter, 25μl Blunt/TA Ligase Master Mix to a 0.2ml PCR tube (15μl End-prepped DNA), spray and mix with a gun, and incubate at 21°C for 25-30min;

2. Add 0.4×beads at room temperature, rotate and mix, and incubate for 5 minutes, then release the supernatant on the magnetic stand;

3. Wash the beads twice with 200μl of freshly prepared 80% alcohol;

4. Add 15μl of nuclease-free water, rotate and mix at room temperature and incubate for 2 minutes, and then put it on the magnetic stand until it is clear;

5. Carefully pipette 13.5μl of supernatant into a 0.2ml PCR tube.

(Four), template amplification

To	Volume (μl)
Platinum SuperFi PCR Master Mix	25
SuperFi GC Enhancer	10
DNA	13.5
RYP-F	0.25
RYP-R	0.25
ABRCD primer	1

2. Prepare the vortex and mix well, and place it on the PCR machine after instant separation to set the program;

3. At the end of the reaction, add 0.6×beads at room temperature, rotate and mix, and incubate for 5 minutes, then release the supernatant on the magnetic stand;

4. Wash the beads twice with 200μl of freshly prepared 80% alcohol;

5. Add 12μl of 10mM Tris-HCl (50mM NaCl) pH 8.0 eluent, rotate and mix at room temperature and incubate for 2min, and then release it on the magnetic stand until it is clear; carefully pipet 11μl of supernatant into a 1.5ml low-adsorption centrifuge tube

6. QC: Take 1μl Qubit for detection.

7. Add 1μl RAP to 10ul of the cleaned and removed product, react at room temperature for 5 minutes, and prepare to go on the machine.

8. According to the standard nanopore sequencing process, 10 cases of different strains were sequenced on a single sample.

Example 5 Detection and analysis of actual samples

Based on the experimental procedure of Example 4, single-sample sequencing was performed on 10 different strains, and it was ensured that each sample was connected to only one barcode. Since only a single barcode is used in each sequencing, if the barcode of the corresponding sample is compared with the barcode that is not corresponding to the sample, it is considered to be a misclassification. The biometric analysis of the present invention uses the official software guppy of Oxford Nanopore Company to evaluate the accuracy of sample classification of the original barcode system, and uses an independent software process to evaluate the accuracy of sample classification of the position-anchored barcode system. The present invention examines the resolving power of 10 groups of position-anchored bar codes, and the final accuracy rate of read classification is statistically calculated within the range of these 10 groups of bar codes.

Results: The results of sample classification using the original barcode system are obviously confused. The accuracy rate of barcode06 classification by guppy in Figure 5 is only 99.954%, of which 0.036% is confused for barcode07, and about 0.01% is confused for other barcodes, with an average accuracy of 99.984% , The confusion rate is as high as 0.016%. This shows that the low accuracy of the single barcode to distinguish the sample can easily cause the "leakage" of high-abundance microorganisms in a sample when multiple samples are on the machine at the same time, which in turn will cause high false positive detections in other samples and mislead subsequent clinical diagnosis and treatment decisions. .

The classification accuracy of the position-anchored barcode system reaches 99.9999% on average, as shown in Figure 5. The reads classified into barcode01, barcode02, barcode05, barcode09 and barcode10 are consistent with the classification accuracy of the simulated data, which are all 100%. Compared with guppy's 99.9% resolution accuracy, it has increased by 3 orders of magnitude, which means that the base for accurately distinguishing samples has increased from a thousand to a million for a single barcode, and the false positive rate caused by misclassification of reads has been reduced by 1000 times. In summary, the classification accuracy of position-anchored barcodes is significantly better than that of single barcodes.

The above description of the specific embodiments of the application does not limit the application, and those skilled in the art can make various changes or modifications according to the application, as long as they do not deviate from the spirit of the application, they shall fall within the scope of the appended claims of the application. .

Claims (15)

1. A position-anchored barcode system for nanopore sequencing library construction, characterized in that the system includes the following structure:

   [BARCODE-ANCHOR] _n -BARCODE _n+1

   Where n≥1,

   The BARCODE is a barcode sequence,

   The ANCHOR is an anchor sequence.
2. The position-anchored barcode system according to claim 1, wherein said 1≤n≤10; preferably, said n is 1, 2 or 3.
3. The position-anchored barcode system of claim 2, wherein the structure is

   FLANK1-[BARCODE-ANCHOR] _n -BARCODE _n+1 -FLANK2,

   The FLANK is a flanking sequence.
4. The position-anchored barcode system according to any one of claims 2 or 3, wherein the BARCODE sequence is the same or different; preferably, the BARCODE sequence is different.
5. The position-anchored barcode system according to any one of claims 2-4, wherein the ANCHOR sequence is the same or different; preferably, the ANCHOR sequence is different.
6. The position-anchored barcode system of any one of claims 2-5, wherein the ANCHOR sequence is 5-50 bp in length; preferably, the ANCHOR sequence is 10-35 bp in length.
7. The position-anchored barcode system according to any one of claims 2-6, wherein the ANCHOR sequence and

   The homology of the BARCODE sequence is less than 70%; preferably, the homology of the ANCHOR sequence and the BARCODE sequence is less than 50%.
8. The position-anchored barcode system according to any one of claims 2-6, wherein the structure is any one of the following:

   FLANK1-BARCODE ₁ -ANCHOR ₁ -BARCODE ₂ -FLANK2;

   FLANK1-BARCODE ₁ -ANCHOR ₁ -BARCODE ₂ -ANCHOR ₂ -BARCODE ₃ -FLANK2;

   Or FLANK1-BARCODE ₁ -ANCHOR ₁ -BARCODE ₂ -ANCHOR ₂ -BARCODE ₃ -ANCHOR ₃ -BARCODE ₅ -FLANK2.
9. The method for preparing a position-anchored barcode system according to any one of claims 1-8, characterized in that: the method comprises directly synthesizing the nucleotide sequence of the position-anchoring barcode system, or linking the preparation site through segmented synthesis. The position anchors the barcode system.
10. A method for constructing a sequencing library, which is characterized in that the position-anchored barcode system according to any one of claims 1-8 is used to construct a sequencing library.
11. A sequencing adapter, characterized in that the sequencing adapter comprises the position-anchored barcode system according to any one of claims 1-8.
12. A composite, characterized in that the composite is connected to the position-anchored barcode system according to any one of claims 1-8.
13. A composition, characterized in that it comprises the position-anchored barcode system according to any one of claims 1-8.
14. A kit for nanopore sequencing library construction, wherein the kit includes the position-anchored barcode system according to any one of claims 1-8, or the sequencing adapter according to claim 11.
15. The application of the position-anchored barcode system according to any one of claims 1-8, wherein the application is any one of the following applications:

    1) Application in improving the classification accuracy of sequencing samples;

    2) Application in reducing false positives in the classification of sequencing samples;

    3) Application in the construction of sequencing library;

    4) Application in sequencing.

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