Improved workflows for high throughput library preparation using the transposome-based nextera system

BMC Biotechnology

Sarah Lamble¹  Elizabeth Batty¹  Moustafa Attar¹  David Buck¹  Rory Bowden¹  Gerton Lunter¹  Derrick Crook²  Bassam El-Fahmawi³  Paolo Piazza¹ 

[1] Wellcome Trust Centre for Human Genetics, OX3 7BN Oxford, UK

[2] Oxford NIHR Biomedical Research Centre, John Radcliffe Hospital, Headley Way, OX3 9DU Oxford, UK

[3] Axygen Inc., A corning Subsidiary, 33120 Central Avenue, 94587 Union City, CA, USA

关键词: Normalisation;    Sequencing;    Library preparation;    High-throughput;    Nextera;   

Others  :  1230603
DOI  :  10.1186/1472-6750-13-104

 received in 2013-03-26, accepted in 2013-10-25,  发布年份 2013

【 摘 要 】

Background

The Nextera protocol, which utilises a transposome based approach to create libraries for Illumina sequencing, requires pure DNA template, an accurate assessment of input concentration and a column clean-up that limits its applicability for high-throughput sample preparation. We addressed the identified limitations to develop a robust workflow that supports both rapid and high-throughput projects also reducing reagent costs.

Results

We show that an initial bead-based normalisation step can remove the need for quantification and improves sample purity. A 75% cost reduction was achieved with a low-volume modified protocol which was tested over genomes with different GC content to demonstrate its robustness. Finally we developed a custom set of index tags and primers which increase the number of samples that can simultaneously be sequenced on a single lane of an Illumina instrument.

Conclusions

We addressed the bottlenecks of Nextera library construction to produce a modified protocol which harnesses the full power of the Nextera kit and allows the reproducible construction of libraries on a high-throughput scale reducing the associated cost of the kit.

【 授权许可】

   
2013 Lamble et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Adey A, Morrison H, Asan G, Xun X, Kitzman J, Turner E, Stackhouse B, MacKenzie A, Caruccio N, Zhang X, Shendure J: Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition. Genome Biol 2010, 11:R119. BioMed Central Full Text
• [2]Parkinson NJ, Maslau S, Ferneyhough B, Zhang G, Gregory L, Buck D, Ragoussis J, Ponting CP, Fischer MD: Preparation of high-quality next-generation sequencing libraries from picogram quantities of target DNA. Genome Res 2012, 22:125-133.
• [3]Caruccio N: Preparation of next-generation sequencing libraries using Nextera technology: simultaneous DNA fragmentation and adaptor tagging by in vitro transposition. Methods Mol Biol 2011, 733:241-255.
• [4]Marine R, Polson SW, Ravel J, Hatfull G, Russell D, Sullivan M, Syed F, Dumas M, Wommack KE: Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA. Appl Environ Microbiol 2011, 77:8071-8079.
• [5]Adey A, Shendure J: Ultra-low-input, tagmentation-based whole-genome bisulfite sequencing. Genome Res 2012, 22:1139-1143.
• [6]Gertz J, Varley KE, Davis NS, Baas BJ, Goryshin IY, Vaidyanathan R, Kuersten S, Myers RM: Transposase mediated construction of RNA-seq libraries. Genome Res 2012, 22:134-141.
• [7]Lazinski DW, Camilli A: Homopolymer tail-mediated ligation PCR: a streamlined and highly efficient method for DNA cloning and library construction. Biotechniques 2013, 54:25-34.
• [8]Didelot X, Eyre D, Cule M, Ip C, Ansari A, Griffiths D, Vaughan A, O’Connor L, Golubchik T, Batty E, et al.: Microevolutionary analysis of Clostridium difficile genomes to investigate transmission. Genome Biol 2012, 13:R118. BioMed Central Full Text
• [9]Oyola S, Otto T, Gu Y, Maslen G, Manske M, Campino S, Turner D, MacInnis B, Kwiatkowski D, Swerdlow H, Quail M: Optimizing illumina next-generation sequencing library preparation for extremely at-biased genomes. BMC Genomics 2012, 13:1. BioMed Central Full Text
• [10]Quail MA, Otto TD, Gu Y, Harris SR, Skelly TF, McQuillan JA, Swerdlow HP, Oyola SO: Optimal enzymes for amplifying sequencing libraries. Nat Methods 2012, 9:10-11.
• [11]Aird D, Ross MG, Chen WS, Danielsson M, Fennell T, Russ C, Jaffe DB, Nusbaum C, Gnirke A: Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol 2011, 12:R18. BioMed Central Full Text
• [12]Goryshin IY, Miller JA, Kil YV, Lanzov VA, Reznikoff WS: Tn5/IS50 target recognition. Proc Natl Acad Sci 1998, 95:10716-10721.
• [13]Reznikoff WS: Tn5 as a model for understanding DNA transposition. Mol Microbiol 2003, 47:1199-1206.
• [14]Lunter G, Goodson M: Stampy: a statistical algorithm for sensitive and fast mapping of Illumina sequence reads. Genome Res 2011, 21:936-939.
• [15]Eyre DW, Golubchik T, Gordon NC, Bowden R, Piazza P, Batty EM, Ip CL, Wilson DJ, Didelot X, O’Connor L, et al.: A pilot study of rapid benchtop sequencing of Staphylococcus aureus and Clostridium difficile for outbreak detection and surveillance. BMJ Open 2012., 2doi:10.1136/bmjopen-2012-001124
• [16]Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Subgroup GPDP: The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009, 25:2078-2079.
• [17]Zerbino DR, Birney E: Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 2008, 18:821-829.
• [18]Darling AE, Tritt A, Eisen JA, Facciotti MT: Mauve assembly metrics. Bioinformatics 2011, 27:2756-2757.
• [19]McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA: The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 2010, 20:1297-1303.