Thermoanaerobacter ethanolicus JW 200 has been identified as a potential sustainable biofuel producer due to its ability to readily ferment carbohydrates to ethanol. A hybrid sequencing approach, combining Oxford Nanopore and Illumina DNA sequence reads, was applied to produce a single contiguous genome sequence of 2,911,280 bp

Ayine, Monica L.

Gill, Steven R.

Gilman, James A.

Hewlett, Mark

James, Paul B.C.

Love, John

Parker, David A.

Power, Ann L.

Singleton, Chloe

Tennant, Richard K.

English

Northumbria University Research Portal

Northumbria Research LinkCitation: Tennant, Richard K., Ayine, Monica L., Power, Ann L., Gilman, James A., Hewlett,  Mark,  James, Paul B.C., Singleton, Chloe, Parker, David A., Love, John and Gill, Steven R. (2019) A Hybrid Sequencing Approach Completes the Genome Sequence of  Thermoanaerobacter ethanolicus JW 200. Microbiology Resource Announcements, 8 (3). e01530-18. ISSN 2576-098X Published by: American Society for MicrobiologyURL: https://doi.org/10.1128/mra.01530-18 <https://doi.org/10.1128/mra.01530-18>This version was downloaded from Northumbria Research Link: http://nrl.northumbria.ac.uk/43267/Northumbria University has developed Northumbria Research Link (NRL) to enable users to access the University’s research output. Copyright © and moral rights for items on NRL are retained by the individual author(s) and/or other copyright owners.  Single copies of full items can be reproduced, displayed or performed, and given to third parties in any format or medium for personal research or study, educational, or not-for-profit purposes without prior permission or charge, provided the authors, title and full bibliographic details are given, as well as a hyperlink and/or URL to the original metadata page. The content must not be changed in any way. Full items must not be sold commercially in any  format or medium without formal permission of the copyright holder.  The full policy is available online: http://nrl.northumbria.ac.uk/pol  i cies.html  This  document  may differ  from the  final,  published version of  the research  and has been made available online in accordance with publisher policies. To read and/or cite from the published version of the research, please visit the publisher’s website (a subscription may be required.)                        A Hybrid Sequencing Approach Completes the GenomeSequence of Thermoanaerobacter ethanolicus JW 200Richard K. Tennant,a Monica L. Ayine,a Ann L. Power,a James A. Gilman,a Mark Hewlett,a Paul B. C. James,aChloe Singleton,a David A. Parker,a John LoveaaBiosciences, College of Environmental and Life Sciences, The BioEconomy Centre, University of Exeter, Exeter, United KingdomABSTRACT Thermoanaerobacter ethanolicus JW 200 has been identified as a poten-tial sustainable biofuel producer due to its ability to readily ferment carbohydratesto ethanol. A hybrid sequencing approach, combining Oxford Nanopore and Illu-mina DNA sequence reads, was applied to produce a single contiguous genome se-quence of 2,911,280 bp.Thermoanaerobacter ethanolicus JW 200 is a thermophilic Gram-positive obligateanaerobe originally isolated from Yellowstone National Park (1). T. ethanolicus JW200 readily ferments glucose to ethanol and has received attention as a sustainablebiofuel producer (2) and for its bioconversion capabilities (3).T. ethanolicus JW 200 was obtained from the DSMZ (DSMZ 2246) and was culturedin Thermoanaerobacter medium (DSMZ 61) at 65°C. DNA was purified in an anaerobiccabinet using a combination of the MP Bio FastDNA spin kit and a high-molecular-weight genomic DNA (gDNA) extraction protocol (4). An overnight culture was resus-pended in CLS-TC buffer (MP Bio, USA) and transferred to a tube containing lysingmatrix A (MP Bio, USA). Lysis tubes were placed into MP Bio FastPrep and homogenizedat 6.0 m·s1 for 40 s. Centrifuged lysates were treated with RNase A, and genomic DNAwas isolated using AMPure XP beads. Purified DNA was quantified using a Qubitfluorometer (Thermo Fisher Scientific, UK), and DNA quality was measured using thegenomic TapeStation assay tapes (Agilent Technologies, USA). Illumina DNA se-quencing libraries were prepared with the Nextera XT library prep kit (Illumina, USA)and sequenced by an Illumina MiSeq instrument, using 150-bp paired-end sequenc-ing. Oxford Nanopore libraries were prepared using the SQK-LSK108 1D genomicDNA ligation kit (Oxford Nanopore, UK) and sequenced on a MinION instrumentusing an R9.4a flow cell (Oxford Nanopore). Oxford Nanopore data were base calledusing Albacore v2.3.3, and DNA sequence data were verified using Porechop (5),yielding 11.37 Gbp of data, with an N50 value of 6,401 bp. Illumina DNA sequencedata were quality controlled and filtered using TrimGalore v0.3.3 (6) with the“paired” parameter selected, which yielded 2,031,855 paired-end reads with an N90value of 138 bp. A hybrid assembly of the Oxford Nanopore and Illumina data wasperformed by MaSuRCA v3.2.3 (7) using the default parameters. The genome wasassembled to a single 2,911,280-bp contiguous sequence with a GC content of34.2% and more than 3,500-fold genome coverage. The assembled genome wasverified by aligning the reads against the de novo assembled genome usingBWA-MEM v0.7.15 (8) with the default parameters. The alignment was visualized inTablet v1.17 (9) to ensure complete coverage. The completed T. ethanolicus genomewas annotated using Prokka v1.12 (10), and 2,818 coding sequences (CDS) wereidentified.Data availability. The complete genome sequence of T. ethanolicus JW 200 isdeposited in GenBank under the accession number CP033580. Illumina and OxfordCitation Tennant RK, Ayine ML, Power AL,Gilman JA, Hewlett M, James PBC, Singleton C,Parker DA, Love J. 2019. A hybrid sequencingapproach completes the genome sequence ofThermoanaerobacter ethanolicus JW 200.Microbiol Resour Announc 8:e01530-18.https://doi.org/10.1128/MRA.01530-18.Editor Steven R. Gill, University of RochesterSchool of Medicine and DentistryCopyright © 2019 Tennant et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.Address correspondence to John Love,J.Love@exeter.ac.uk.Received 13 November 2018Accepted 10 December 2018Published 17 January 2019GENOME SEQUENCEScrossmVolume 8 Issue 3 e01530-18 mra.asm.org 1 on May 28, 2020 by guesthttp://mra.asm.org/Downloaded from Nanopore DNA sequence reads have been deposited in the NCBI Sequence ReadArchive (accession numbers SRR8113455 and SRR8113456).ACKNOWLEDGMENTSThis work was supported by a grant from Shell Research Ltd.We acknowledge the Exeter Sequencing Service for its assistance in sequencing theIllumina libraries.We declare no competing interests.REFERENCES1. Wiegel J, Ljungdahl LG. 1981. Thermoanaerobacter ethanolicus gen. nov.,spec. nov., a new, extreme thermophilic, anaerobic bacterium. ArchMicrobiol 128:343–348. https://doi.org/10.1007/BF00405910.2. Shao X, Zhou J, Olson DG, Lynd LR. 2016. A markerless gene deletion andintegration system for Thermoanaerobacter ethanolicus. Biotechnol Bio-fuels 9:100. https://doi.org/10.1186/s13068-016-0514-1.3. Hild HM, Stuckey DC, Leak DJ. 2003. Effect of nutrient limitation onproduct formation during continuous fermentation of xylose with Ther-moanaerobacter ethanolicus JW200 Fe(7). Appl Microbiol Biotechnol 60:679–686. https://doi.org/10.1007/s00253-002-1175-5.4. Mayjonade B, Gouzy J, Donnadieu C, Pouilly N, Marande W, Callot C,Langlade N, Muños S. 2016. Extraction of high-molecular-weightgenomic DNA for long-read sequencing of single molecules. Biotech-niques 61:203–205. https://doi.org/10.2144/000114460.5. Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Completing bacterial ge-nome assemblies with multiplex MinION sequencing. Microb Genom3:e000132. https://doi.org/10.1099/mgen.0.000132.6. Kruegar F. 2014. Trim Galore! Babraham Bioinformatics, Cambridge,United Kingdom. https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/.7. Zimin AV, Marçais G, Puiu D, Roberts M, Salzberg SL, Yorke JA. 2013. TheMaSuRCA genome assembler. Bioinformatics 29:2669–2677. https://doi.org/10.1093/bioinformatics/btt476.8. Li H, Durbin R. 2010. Fast and accurate long-read alignment withBurrows-Wheeler transform. Bioinformatics 26:589–595. https://doi.org/10.1093/bioinformatics/btp698.9. Milne I, Bayer M, Stephen G, Cardle L, Marshall D. 2016. Tablet: visual-izing next-generation sequence assemblies and mappings. Methods MolBiol 1374:253–268. https://doi.org/10.1007/978-1-4939-3167-5_14.10. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioin-formatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu153.Tennant et al.Volume 8 Issue 3 e01530-18 mra.asm.org 2 on May 28, 2020 by guesthttp://mra.asm.org/Downloaded from 

A Hybrid Sequencing Approach Completes the Genome Sequence of Thermoanaerobacter ethanolicus JW 200

http://nrl.northumbria.ac.uk/43267/1/Microbiology%20Resource%20Announcements-2019-Tennant-e01530-18.full.pdf

A Hybrid Sequencing Approach Completes the Genome Sequence of Thermoanaerobacter ethanolicus JW 200

Abstract

Similar works

Full text

Available Versions

Northumbria University Research Portal