BACKGROUND: There has been an exponential growth in the number of genome sequencing projects since the introduction of next generation DNA sequencing technologies. Genome projects have increasingly involved assembly of whole genome data which produces inferior assemblies compared to traditional Sanger sequencing of genomic fragments cloned into bacterial artificial chromosomes (BACs). While whole genome shotgun sequencing using next generation sequencing (NGS) is relatively fast and inexpensive, this method is extremely challenging for highly complex genomes, where polyploidy or high repeat content confounds accurate assembly, or where a highly accurate 'gold' reference is required. Several attempts have been made to improve genome sequencing approaches by incorporating NGS methods, to variable success. RESULTS: We present the application of a novel BAC sequencing approach which combines indexed pools of BACs, Illumina paired read sequencing, a sequence assembler specifically designed for complex BAC assembly, and a custom bioinformatics pipeline. We demonstrate this method by sequencing and assembling BAC cloned fragments from bread wheat and sugarcane genomes. CONCLUSIONS: We demonstrate that our assembly approach is accurate, robust, cost effective and scalable, with applications for complete genome sequencing in large and complex genomes.

CSIRO Plant Industry Brisbane QLD 4072 Australia

Institute of Experimental Botany Centre of the Region Haná for Biotechnological and Agricultural Research Šlechtitelů 31 78371 Olomouc Czech Republic

School of Agriculture and Food Science University of Queensland Brisbane QLD 4072 Australia

School of Agriculture and Food Science University of Queensland Brisbane QLD 4072 Australia ; Centre for Biotechnology and Bioinformatics College of Biological and Physical Sciences University of Nairobi P O Box 30197 Nairobi 00100 Kenya

School of Agriculture and Food Science University of Queensland Brisbane QLD 4072 Australia ; School of Plant Biology University of Western Australia Perth WA 6009 Australia

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Ruperao P, Chan C-KK, Azam S, Karafiátová M, Hayashi S, Čížková J, Saxena RK, Šimková H, Song C, Vrána J, Chitikineni A, Visendi P, Gaur PM, Millán T, Singh KB, Tara B, Wang J, Batley J, Doležel J, Varshney RK, Edwards D. A chromosomal genomics approach to assess and validate the desi and kabuli draft chickpea genome assemblies. Plant Biotechnol J. 2014;12:778–786. doi: 10.1111/pbi.12182. PubMed DOI

Kelley DR, Salzberg SL. Detection and correction of false segmental duplications caused by genome mis-assembly. Genome Biol. 2010;11:R28. doi: 10.1186/gb-2010-11-3-r28. PubMed DOI PMC

Alkan C, Sajjadian S, Eichler EE. Limitations of next-generation genome sequence assembly. Nat Methods. 2010;8:61–65. doi: 10.1038/nmeth.1527. PubMed DOI PMC

Berkman PJ, Visendi P, Lee HC, Stiller J, Manoli S, Lorenc MT, Lai K, Batley J, Fleury D, Šimková H, Kubaláková M, Weining S, Doležel J, Edwards D. Dispersion and domestication shaped the genome of bread wheat. Plant Biotechnol J. 2013;11:564–571. doi: 10.1111/pbi.12044. PubMed DOI

Kajitani R, Toshimoto K, Noguchi H, Toyoda A, Ogura Y, Okuno M, Yabana M, Harada M, Nagayasu E, Maruyama H, Kohara Y, Fujiyama A, Hayashi T, Itoh T. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Res. 2014;24:1384–1395. doi: 10.1101/gr.170720.113. PubMed DOI PMC

Paterson AH, Bowers JE, Bruggmann R, Dubchak I, Grimwood J, Gundlach H, Haberer G, Hellsten U, Mitros T, Poliakov A, Schmutz J, Spannagl M, Tang H, Wang X, Wicker T, Bharti AK, Chapman J, Feltus FA, Gowik U, Grigoriev IV, Lyons E, Maher CA, Martis M, Narechania A, Otillar RP, Penning BW, Salamov AA, Wang Y, Zhang L, Carpita NC, et al. The sorghum bicolor genome and the diversification of grasses. Nature. 2009;457:551–556. doi: 10.1038/nature07723. PubMed DOI

Au KF, Underwood JG, Lee L, Wong WH. Improving PacBio long read accuracy by short read alignment. PLoS One. 2012;7:e46679. doi: 10.1371/journal.pone.0046679. PubMed DOI PMC

Salzberg SL, Phillippy AM, Zimin A, Puiu D, Magoc T, Koren S, Treangen TJ, Schatz MC, Delcher AL, Roberts M, Marcais G, Pop M, Yorke JA. GAGE: a critical evaluation of genome assemblies and assembly algorithms. Genome Res. 2012;22:557–567. doi: 10.1101/gr.131383.111. PubMed DOI PMC

Chen M, Presting G, Barbazuk WB, Goicoechea JL, Blackmon B, Fang G, Kim H, Frisch D, Yu Y, Sun S, Higingbottom S, Phimphilai J, Phimphilai D, Thurmond S, Gaudette B, Li P, Liu J, Hatfield J, Main D, Farrar K, Henderson C, Barnett L, Costa R, Williams B, Walser S, Atkins M, Hall C, Budiman MA, Tomkins JP, Luo M, et al. An integrated physical and genetic map of the rice genome. Plant Cell. 2002;14:537–545. doi: 10.1105/tpc.010485. PubMed DOI PMC

Choi SR, Teakle GR, Plaha P, Kim JH, Allender CJ, Beynon E, Piao ZY, Soengas P, Han TH, King GJ, Barker GC, Hand P, Lydiate DJ, Batley J, Edwards D, Koo DH, Bang JW, Park B-S, Lim YP. The reference genetic linkage map for the multinational Brassica rapa genome sequencing project. Theor Appl Genet. 2007;115:777–792. doi: 10.1007/s00122-007-0608-z. PubMed DOI

Brenchley R, Spannagl M, Pfeifer M, Barker GLA, D’Amore R, Allen AM, McKenzie N, Kramer M, Kerhornou A, Bolser D, Kay S, Waite D, Trick M, Bancroft I, Gu Y, Huo N, Luo M-C, Sehgal S, Gill B, Kianian S, Anderson O, Kersey P, Dvorak J, McCombie WR, Hall A, Mayer KFX, Edwards KJ, Bevan MW, Hall N. Analysis of the bread wheat genome using whole-genome shotgun sequencing. Nature. 2012;491:705–710. doi: 10.1038/nature11650. PubMed DOI PMC

Poland JA, Brown PJ, Sorrells ME, Jannink J-L. Development of high-density genetic maps for barley and wheat using a novel two-enzyme genotyping-by-sequencing approach. PLoS One. 2012;7:e32253. doi: 10.1371/journal.pone.0032253. PubMed DOI PMC

Mascher M, Muehlbauer GJ, Rokhsar DS, Chapman J, Schmutz J, Barry K, Muñoz-Amatriaín M, Close TJ, Wise RP, Schulman AH, Himmelbach A, Mayer KFX, Scholz U, Poland JA, Stein N, Waugh R. Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ) Plant J. 2013;76:718–727. doi: 10.1111/tpj.12319. PubMed DOI PMC

Chandler VL. The maize genome sequencing project. Plant Physiol. 2002;130:1594–1597. doi: 10.1104/pp.015594. PubMed DOI PMC

Martienssen RA, Rabinowicz PD, O’Shaughnessy A, McCombie WR. Sequencing the maize genome. Curr Opin Plant Biol. 2004;7:102–107. doi: 10.1016/j.pbi.2004.01.010. PubMed DOI

Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S, Liang C, Zhang J, Fulton L, Graves TA, Minx P, Reily AD, Courtney L, Kruchowski SS, Tomlinson C, Strong C, Delehaunty K, Fronick C, Courtney B, Rock SM, Belter E, Du F, Kim K, Abbott RM, Cotton M, Levy A, Marchetto P, Ochoa K, Jackson SM, Gillam B, et al. The B73 maize genome: complexity, diversity, and dynamics. Science. 2009;326:1112–1115. doi: 10.1126/science.1178534. PubMed DOI

Eilam T, Anikster Y, Millet E, Manisterski J, Sagi-Assif O, Feldman M. Genome size and genome evolution in diploid Triticeae species. Genome. 2007;50:1029–1037. doi: 10.1139/G07-083. PubMed DOI

Wicker T, Mayer KFX, Gundlach H, Martis M, Steuernagel B, Scholz U, Šimková H, Kubaláková M, Choulet F, Taudien S, Platzer M, Feuillet C, Fahima T, Budak H, Doležel J, Keller B, Stein N. Frequent gene movement and pseudogene evolution is common to the large and complex genomes of wheat, barley, and their relatives. Plant Cell. 2011;23:1706–1718. doi: 10.1105/tpc.111.086629. PubMed DOI PMC

D’Hont A. Unraveling the genome structure of polyploids using FISH and GISH; examples of sugarcane and banana. Cytogenet Genome Res. 2005;109:27–33. doi: 10.1159/000082378. PubMed DOI

Tomkins JP, Yu Y, Miller-Smith H, Frisch DA, Woo SS, Wing RA. A bacterial artificial chromosome library for sugarcane. Theor Appl Genet. 1999;99:419–424. doi: 10.1007/s001220051252. PubMed DOI

Kazakoff SH, Imelfort M, Edwards D, Koehorst J, Biswas B, Batley J, Scott PT, Gresshoff PM. Capturing the biofuel wellhead and powerhouse: the chloroplast and mitochondrial genomes of the leguminous feedstock tree Pongamia pinnata. PLoS One. 2011;7:e51687–e51687. doi: 10.1371/journal.pone.0051687. PubMed DOI PMC

Šimková H, Safár J, Kubaláková M, Suchánková P, Cíhalíková J, Robert-Quatre H, Azhaguvel P, Weng Y, Peng J, Lapitan NLV, Ma Y, You FM, Luo M-C, Bartos J, Doležel J. BAC libraries from wheat chromosome 7D: efficient tool for positional cloning of aphid resistance genes. J Biomed Biotechnol. 2011;2011:1–11. doi: 10.1155/2011/302543. PubMed DOI PMC

Park N, Shirley L, Gu Y, Keane TM, Swerdlow H, Quail MA. An improved approach to mate-paired library preparation for Illumina sequencing. Methods Next Gen Seq. 2013;1:10–20.

Taudien S, Steuernagel B, Ariyadasa R, Schulte D, Schmutzer T, Groth M, Felder M, Petzold A, Scholz U, Mayer KF, Stein N, Platzer M. Sequencing of BAC pools by different next generation sequencing platforms and strategies. BMC Res Notes. 2011;4:411. doi: 10.1186/1756-0500-4-411. PubMed DOI PMC

Luo M-C, Thomas C, You FM, Hsiao J, Ouyang S, Buell CR, Malandro M, McGuire PE, Anderson OD, Dvorak J. High-throughput fingerprinting of bacterial artificial chromosomes using the snapshot labeling kit and sizing of restriction fragments by capillary electrophoresis. Genomics. 2003;82:378–389. doi: 10.1016/S0888-7543(03)00128-9. PubMed DOI

Soderlund C, Humphray S, Dunham A, French L. Contigs built with fingerprints, markers, and FPC V4.7. Genome Res. 2000;10:1772–1787. doi: 10.1101/gr.GR-1375R. PubMed DOI PMC

Zhang H-B, Choi S, Woo S-S, Li Z, Wing R. Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population. Mol Breeding. 1996;2:11–24. doi: 10.1007/BF00171348. DOI

Tomkins JP, Yu Y, Miller-Smith H, Frisch DA, Woo SS, Wing RA. A bacterial artificial chromosome library for sugarcane. Theor Appl Genet. 1999;99:419–424. doi: 10.1007/s001220051252. PubMed DOI

Cleveland WS, Devlin SJ. Locally weighted regression: an approach to regression analysis by local fitting. J Am Stat Assoc. 1988;83:596–610. doi: 10.1080/01621459.1988.10478639. DOI

McGinnis S, Madden TL. BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Res. 2004;32(Web Server):W20–W25. doi: 10.1093/nar/gkh435. PubMed DOI PMC

Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL. Versatile and open software for comparing large genomes. Genome Biol. 2004;5:R12. doi: 10.1186/gb-2004-5-2-r12. PubMed DOI PMC

Yin T, Cook D, Lawrence M. ggbio: an R package for extending the grammar of graphics for genomic data. Genome Biol. 2012;13:R77. doi: 10.1186/gb-2012-13-8-r77. PubMed DOI PMC

Li R, Yu C, Li Y, Lam TW, Yiu SM, Kristiansen K, Wang J. SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics. 2009;25:1966–1967. doi: 10.1093/bioinformatics/btp336. PubMed DOI

Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011;17:10. doi: 10.14806/ej.17.1.200. DOI

O’Connell J, Schulz-Trieglaff O, Carlson E, Hims MM, Gormley NA, Cox AJ. NxTrim: optimized trimming of Illumina mate pair reads. bioRxiv 2014;007666. PubMed

Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics. 2011;27:578–579. doi: 10.1093/bioinformatics/btq683. PubMed DOI