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Sequencing artifacts derived from a library preparation method using enzymatic fragmentation.

ABSTRACT: DNA fragmentation is a fundamental step during library preparation in hybridization capture-based, short-read sequencing. Ultra-sonication has been used thus far to prepare DNA of an appropriate size, but this method is associated with a considerable loss of DNA sample. More recently, studies have employed library preparation methods that rely on enzymatic fragmentation with DNA endonucleases to minimize DNA loss, particularly in nano-quantity samples. Yet, despite their wide use, the effect of enzymatic fragmentation on the resultant sequences has not been carefully assessed. Here, we used pairwise comparisons of somatic variants of the same tumor DNA samples prepared using ultrasonic and enzymatic fragmentation methods. Our analysis revealed a substantially larger number of recurrent artifactual SNVs/indels in endonuclease-treated libraries as compared with those created through ultrasonication. These artifacts were marked by palindromic structure in the genomic context, positional bias in sequenced reads, and multi-nucleotide substitutions. Taking advantage of these distinctive features, we developed a filtering algorithm to distinguish genuine somatic mutations from artifactual noise with high specificity and sensitivity. Noise cancelling recovered the composition of the mutational signatures in the tumor samples. Thus, we provide an informatics algorithm as a solution to the sequencing errors produced as a consequence of endonuclease-mediated fragmentation, highlighted for the first time in this study.

SUBMITTER: Tanaka N

PROVIDER: S-EPMC6941819 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Sequencing artifacts derived from a library preparation method using enzymatic fragmentation.

Tanaka Norio N Takahara Akihisa A Hagio Taichi T Nishiko Rika R Kanayama Junko J Gotoh Osamu O Mori Seiichi S

PloS one 20200103 1

DNA fragmentation is a fundamental step during library preparation in hybridization capture-based, short-read sequencing. Ultra-sonication has been used thus far to prepare DNA of an appropriate size, but this method is associated with a considerable loss of DNA sample. More recently, studies have employed library preparation methods that rely on enzymatic fragmentation with DNA endonucleases to minimize DNA loss, particularly in nano-quantity samples. Yet, despite their wide use, the effect of ...[more]

PMID: 31899787

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Project description:BackgroundNovel commercial kits for whole genome library preparation for next-generation sequencing on Illumina platforms promise shorter workflows, lower inputs and cost savings. Time savings are achieved by employing enzymatic DNA fragmentation and by combining end-repair and tailing reactions. Fewer cleanup steps also allow greater DNA input flexibility (1 ng-1 μg), PCR-free options from 100 ng DNA, and lower price as compared to the well-established sonication and tagmentation-based DNA library preparation kits.ResultsWe compared the performance of four enzymatic fragmentation-based DNA library preparation kits (from New England Biolabs, Roche, Swift Biosciences and Quantabio) to a tagmentation-based kit (Illumina) using low input DNA amounts (10 ng) and PCR-free reactions with 100 ng DNA. With four technical replicates of each input amount and kit, we compared the kits' fragmentation sequence-bias as well as performance parameters such as sequence coverage and the clinically relevant detection of single nucleotide and indel variants. While all kits produced high quality sequence data and demonstrated similar performance, several enzymatic fragmentation methods produced library insert sizes which deviated from those intended. Libraries with longer insert lengths performed better in terms of coverage, SNV and indel detection. Lower performance of shorter-insert libraries could be explained by loss of sequence coverage to overlapping paired-end reads, exacerbated by the preferential sequencing of shorter fragments on Illumina sequencers. We also observed that libraries prepared with minimal or no PCR performed best with regard to indel detection.ConclusionsThe enzymatic fragmentation-based DNA library preparation kits from NEB, Roche, Swift and Quantabio are good alternatives to the tagmentation based Nextera DNA flex kit from Illumina, offering reproducible results using flexible DNA inputs, quick workflows and lower prices. Libraries with insert DNA fragments longer than the cumulative sum of both read lengths avoid read overlap, thus produce more informative data that leads to strongly improved genome coverage and consequently also increased sensitivity and precision of SNP and indel detection. In order to best utilize such enzymatic fragmentation reagents, researchers should be prepared to invest time to optimize fragmentation conditions for their particular samples.

Project description:BackgroundHybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors.ResultsWe used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation "blacklist" in the BED region to reduce errors in downstream analyses.ConclusionsWe first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.

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Sequencing artifacts derived from a library preparation method using enzymatic fragmentation.

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Sequencing artifacts derived from a library preparation method using enzymatic fragmentation.

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