Item request has been placed! ×
Item request cannot be made. ×
loading  Processing Request

Optimization of enzymatic fragmentation is crucial to maximize genome coverage: a comparison of library preparation methods for Illumina sequencing.

Item request has been placed! ×
Item request cannot be made. ×
loading   Processing Request
  • Additional Information
    • Source:
      Publisher: BioMed Central Country of Publication: England NLM ID: 100965258 Publication Model: Electronic Cited Medium: Internet ISSN: 1471-2164 (Electronic) Linking ISSN: 14712164 NLM ISO Abbreviation: BMC Genomics Subsets: MEDLINE
    • Publication Information:
      Original Publication: London : BioMed Central, [2000-
    • Subject Terms:
    • Abstract:
      Background: Novel 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.
      Results: We 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.
      Conclusions: The 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.
      (© 2022. The Author(s).)
    • References:
      Semin Cancer Biol. 2021 Jun 25;:. (PMID: 34175442)
      Bioinformatics. 2009 Jul 15;25(14):1754-60. (PMID: 19451168)
      Genome Biol. 2019 Apr 29;20(1):85. (PMID: 31036053)
      Nucleic Acids Res. 2012 May;40(10):e72. (PMID: 22323520)
      Forensic Sci Int Genet. 2017 Jul;29:174-180. (PMID: 28448897)
      Genomics. 1988 Apr;2(3):231-9. (PMID: 3294162)
      IEEE Trans Vis Comput Graph. 2014 Dec;20(12):1983-92. (PMID: 26356912)
      PLoS One. 2011;6(11):e28240. (PMID: 22140562)
      Curr Protoc Bioinformatics. 2013;43:11.10.1-11.10.33. (PMID: 25431634)
      Semin Cancer Biol. 2021 Jun 10;:. (PMID: 34119643)
      BMC Res Notes. 2016 May 12;9:269. (PMID: 27176120)
      Nucleic Acids Res. 2016 Jul 8;44(W1):W160-5. (PMID: 27079975)
      BMC Genomics. 2018 Oct 1;19(1):722. (PMID: 30285621)
      Sci Rep. 2019 Feb 27;9(1):2856. (PMID: 30814542)
      BMC Genomics. 2016 May 25;17:399. (PMID: 27225215)
      Nat Biotechnol. 2014 Mar;32(3):246-51. (PMID: 24531798)
      DNA Res. 2019 Oct 1;26(5):391-398. (PMID: 31364694)
      Genome Res. 2017 Jan;27(1):157-164. (PMID: 27903644)
      Nat Methods. 2020 Mar;17(3):261-272. (PMID: 32015543)
      Genome Med. 2020 Oct 26;12(1):91. (PMID: 33106175)
      Gigascience. 2021 Feb 16;10(2):. (PMID: 33590861)
      Genome Med. 2014 Oct 28;6(10):89. (PMID: 25426171)
      Nucleic Acids Res. 2013 Apr 1;41(6):e67. (PMID: 23303777)
      Nat Protoc. 2020 Aug;15(8):2279-2300. (PMID: 32612278)
      Genome Biol. 2010;11(12):R119. (PMID: 21143862)
      Dialogues Clin Neurosci. 2016 Sep;18(3):299-312. (PMID: 27757064)
      PLoS One. 2020 Jan 3;15(1):e0227427. (PMID: 31899787)
      Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10716-21. (PMID: 9724770)
      Mol Ecol Resour. 2016 Mar;16(2):511-23. (PMID: 26345272)
      BMC Genomics. 2015 Aug 28;16:648. (PMID: 26315384)
      Nat Genet. 2014 Dec;46(12):1343-9. (PMID: 25326703)
      Sci Data. 2016 Jun 07;3:160025. (PMID: 27271295)
    • Contributed Indexing:
      Keywords: Next Generation Sequencing; Whole genome sequencing; enzymatic fragmentation; insert size; library preparation
    • Publication Date:
      Date Created: 20220202 Date Completed: 20220203 Latest Revision: 20220205
    • Publication Date:
    • Accession Number:
    • Accession Number:
    • Accession Number: