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Homology-based enzymatic DNA fragment assembly-based illumina sequencing library preparation.

ABSTRACT: The current Illumina HiSeq and MiSeq platforms can generate paired-end reads of up to 2 x 250?bp and 2 x 300?bp in length, respectively. These read lengths may be substantially longer than genomic regions of interest when a DNA sequencing library is prepared through a target enrichment-based approach. A sequencing library preparation method has been developed based on the homology-based enzymatic DNA fragment assembly scheme to allow processing of multiple PCR products within a single read. Target sequences were amplified using locus-specific PCR primers with 8?bp tags, and using the tags, homology-based enzymatic DNA assembly was performed with DNA polymerase, T7 exonuclease and T4 DNA ligase. Short PCR amplicons can hence be assembled into a single molecule, along with sequencing adapters specific to the Illumina platforms. As a proof-of-concept experiment, short PCR amplicons (57-66?bp in length) derived from genomic DNA templates of field pea and containing variable nucleotide locations were assembled and sequenced on the MiSeq platform. The results were validated with other genotyping methods. When 5 PCR amplicons were assembled, 4.3 targeted sequences (single-nucleotide polymorphisms) on average were successfully identified within each read. The utility of this for sequencing of short fragments has consequently been demonstrated.

SUBMITTER: Shinozuka H

PROVIDER: S-EPMC6994068 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Homology-based enzymatic DNA fragment assembly-based illumina sequencing library preparation.

Shinozuka Hiroshi H Sudheesh Shimna S Shinozuka Maiko M Cogan Noel O I NOI

Biology methods & protocols 20180227 1

The current Illumina HiSeq and MiSeq platforms can generate paired-end reads of up to 2 x 250 bp and 2 x 300 bp in length, respectively. These read lengths may be substantially longer than genomic regions of interest when a DNA sequencing library is prepared through a target enrichment-based approach. A sequencing library preparation method has been developed based on the homology-based enzymatic DNA fragment assembly scheme to allow processing of multiple PCR products within a single read. Targ ...[more]

PMID: 32161795

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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:BackgroundMassively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences.ResultsWe have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates.ConclusionWe have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.

Project description:BackgroundNext-generation sequencing (NGS) technology has paved the way for rapid and cost-efficient de novo sequencing of bacterial genomes. In particular, the introduction of PCR-free library preparation procedures (LPPs) lead to major improvements as PCR bias is largely reduced. However, in order to facilitate the assembly of Illumina paired-end sequence data and to enhance assembly performance, an increase of insert sizes to facilitate the repeat bridging and resolution capabilities of current state of the art assembly tools is needed. In addition, information concerning the relationships between genomic GC content, library insert size and sequencing quality as well as the influence of library insert size, read length and sequencing depth on assembly performance would be helpful to specifically target sequencing projects.ResultsOptimized DNA fragmentation settings and fine-tuned resuspension buffer to bead buffer ratios during fragment size selection were integrated in the Illumina TruSeq(®) DNA PCR-free LPP in order to produce sequencing libraries varying in average insert size for bacterial genomes within a range of 35.4-73.0 % GC content. The modified protocol consumes only half of the reagents per sample, thus doubling the number of preparations possible with a kit. Examination of different libraries revealed that sequencing quality decreases with increased genomic GC content and with larger insert sizes. The estimation of assembly performance using assembly metrics like corrected NG50 and NGA50 showed that libraries with larger insert sizes can result in substantial assembly improvements as long as appropriate assembly tools are chosen. However, such improvements seem to be limited to genomes with a low to medium GC content. A positive trend between read length and assembly performance was observed while sequencing depth is less important, provided a minimum coverage is reached.ConclusionsBased on the optimized protocol developed, sequencing libraries with flexible insert sizes and lower reagent costs can be generated. Furthermore, increased knowledge about the interplay of sequencing quality, insert size, genomic GC content, read length, sequencing depth and the assembler used will help molecular biologists to set up an optimal experimental and analytical framework with respect to Illumina next-generation sequencing of bacterial genomes.

Dataset Information

Homology-based enzymatic DNA fragment assembly-based illumina sequencing library preparation.

Publications

Homology-based enzymatic DNA fragment assembly-based illumina sequencing library preparation.

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