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Assembling the genome sequences of the plastid and mitochondrion of white spruce

45216dabbd95ea1087fa68234932eb62?s=47 Shaun Jackman
January 14, 2014

Assembling the genome sequences of the plastid and mitochondrion of white spruce

The genome sequences of the plastid and mitochondrion of white spruce (Picea glauca) are assembled from whole genome Illumina sequencing data using ABySS and aligned to the Norway spruce (Picea abies) using BWA-MEM. The putative mitochondrial sequences are classified using k-means clustering in R. The plastid genome is 120 kbp and the putative mitochondrial genome is 6 Mbp.

45216dabbd95ea1087fa68234932eb62?s=128

Shaun Jackman

January 14, 2014
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  1. Assembling the genome sequences of the plastid and mitochondrion of

    white spruce PAG 2014 Bioinformatics Workshop Shaun Jackman @sjackman 2014-01-14 1 Shaun D Jackman1, Anthony Raymond1, Ben Vandervalk1, Hamid Mohamadi1, René Warren1, Stephen Pleasance1,
 Robin Coope1, Macaire MS Yuen2, Christopher Keeling2, Carol Ritland2, Jean Bousquet3, Alvin Yanchuk4,
 Kermit Ritland2, John MacKay3, Steven JM Jones1, Jörg C Bohlmann2 and İnanç Birol1 (1) BC Cancer Agency, Genome Sciences Centre, Vancouver, BC, Canada, (2) University of British Columbia, Vancouver, BC, Canada,
 (3) Univesité Laval, Quebec, QC, Canada, (4) British Columbia Ministry of Forests, Victoria, BC, Canada Photo credit: Joseph O'Brien, USDA Forest Service, bugwood.org
  2. Assembling the Genome Sequences of the Plastid and Mitochondrion of

    White Spruce (Picea glauca) PAG 2014 Bioinformatics Workshop Shaun Jackman @sjackman 2014-01-14 2
  3. 10.1101/gr.089532.108 Access the most recent version at doi: 2009 19:

    1117-1123 originally published online February 27, 2009 Genome Res. Jared T. Simpson, Kim Wong, Shaun D. Jackman, et al. ABySS: A parallel assembler for short read sequence data Material Supplemental http://genome.cshlp.org/content/suppl/2009/04/27/gr.089532.108.DC1.html References http://genome.cshlp.org/content/19/6/1117.full.html#related-urls Article cited in: http://genome.cshlp.org/content/19/6/1117.full.html#ref-list-1 This article cites 31 articles, 14 of which can be accessed free at: Open Access Freely available online through the Genome Research Open Access option. Related Content Genome Res. December 7, 2011 : Jared T Simpson and Richard Durbin structures Efficient de novo assembly of large genomes using compressed data Genome Res. December 6, 2011 : Steven L Salzberg, Adam M Phillippy, Aleksey V Zimin, et al. GAGE: A critical evaluation of genome assemblies and assembly algorithms service Email alerting click here top right corner of the article or Receive free email alerts when new articles cite this article - sign up in the box at th Cold Spring Harbor Laboratory Press on January 4, 2012 - Published by genome.cshlp.org Downloaded from ARTICLE OPEN doi:10.1038/nature12211 The Norway spruce genome sequence and conifer genome evolution Lists of authors and their affiliations appear at the end of the paper Conifers have dominated forests for more than 200 million years and are of huge ecological and economic importance. Here we present the draft assembly of the 20-gigabase genome of Norway spruce (Picea abies), the first available for any gymnosperm. The number of well-supported genes (28,354) is similar to the .100 times smaller genome of Arabidopsis thaliana, and there is no evidence of a recent whole-genome duplication in the gymnosperm lineage. Instead, the large genome size seems to result from the slow and steady accumulation of a diverse set of long-terminal repeat transposable elements, possibly owing to the lack of an efficient elimination mechanism. Comparative sequencing of Pinus sylvestris, Abies sibirica, Juniperus communis, Taxus baccata and Gnetum gnemon reveals that the transposable element diversity is shared among extant conifers. Expression of 24-nucleotide small RNAs, previously implicated in transposable element silencing, is tissue-specific and much lower than in other plants. We further identify numerous long (.10,000 base pairs) introns, gene-like fragments, uncharacterized long non-coding RNAs and short RNAs. This opens up new genomic avenues for conifer forestry and breeding. Gymnosperms are a group of land plants comprising the extant taxa, cycads,Ginkgo, gnetophytes and conifers. Gymnospermsfirst appeared more than300 million years ago (Myrago)1, wellbefore theangiosperm lineage separated from the stem group of extant gymnosperms2. The negates the production of inbred lines that could facilitate genome assembly. The availability of conifer genome sequences would enable com- parative analyses of genome architecture and the evolution of key Vol. 29 no. 12 2013, pages 1492–1497 BIOINFORMATICS ORIGINAL PAPER doi:10.1093/bioinformatics/btt178 Genome analysis Advance Access publication May 22, 2013 Assembling the 20 Gb white spruce (Picea glauca) genome from whole-genome shotgun sequencing data Inanc Birol1,2,3,*, Anthony Raymond1, Shaun D. Jackman1, Stephen Pleasance1, Robin Coope1, Greg A. Taylor1, Macaire Man Saint Yuen4, Christopher I. Keeling4, Dana Brand1, Benjamin P. Vandervalk1, Heather Kirk1, Pawan Pandoh1, Richard A. Moore1, Yongjun Zhao1, Andrew J. Mungall1, Barry Jaquish5, Alvin Yanchuk5, Carol Ritland4,6, Brian Boyle7, Jean Bousquet7,8, Kermit Ritland6, John MacKay7,8, Jo ¨ rg Bohlmann4,6 and Steven J.M. Jones1,2,9 1Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC V5Z 4S6, Canada, 2Department of Medical Genetics, University of British Columbia, Vancouver, BC V6H 3N1, Canada, 3School of Computing Science, Simon Fraser University, Burnaby, BC V5A 1S6, Canada, 4Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada, 5British Columbia Ministry of Forests, Lands and Natural Resource Operations, Victoria, BC V8W 9C2, Canada, 6Department of Forest Sciences, University of British Columbia, Vancouver, BC V6T 1Z4, Canada, 7Institute for Systems and Integrative Biology, Universite ´ Laval, Que ´ bec, QC G1K 7P4, Canada, 8Department of Wood and Forest Sciences, Universite ´ Laval, Que ´ bec, QC G1V 0A6, Canada and 9Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada Associate Editor: Michael Brudno ABSTRACT White spruce (Picea glauca) is a dominant conifer of the boreal forests of North America, and providing genomics resources for this commer- cially valuable tree will help improve forest management and conser- vation efforts. Sequencing and assembling the large and highly repetitive spruce genome though pushes the boundaries of the current technology. Here, we describe a whole-genome shotgun sequencing strategy using two Illumina sequencing platforms and an assembly approach using the ABySS software. We report a 20.8 giga base pairs draft genome in 4.9 million scaffolds, with a scaffold N50 of 20356bp. We demonstrate how recent improvements in the sequen- cing technology, especially increasing read lengths and paired end reads from longer fragments have a major impact on the assembly contiguity. We also note that scalable bioinformatics tools are instru- mental in providing rapid draft assemblies. Availability: The Picea glauca genome sequencing and assembly data are available through NCBI (Accession#: ALWZ0100000000 PID: PRJNA83435). http://www.ncbi.nlm.nih.gov/bioproject/83435. Contact: ibirol@bcgsc.ca Supplementary information: Supplementary data are available at Bioinformatics online. Received on March 20, 2013; revised on April 10, 2013; accepted on April 11, 2013 1 INTRODUCTION The assembly of short reads to develop genomic resources for non-model species remains an active area of development (Schatz et al., 2012). The feasibility of the approach and its scalability to large genomes was demonstrated by the ABySS publication (Simpson et al., 2009) using human genome sequencing data and was later used to assemble the panda genome with the SOAPdenovo tool (Li et al., 2010). The technology provides high quality results, as demonstrated for bacteria (Bankevich et al., 2012; Ladner et al., 2013; Ribeiro et al., 2012), and has been successfully applied numerous times on more complex gen- omes (Chan et al., 2011; Chu et al., 2011; Diguistini et al., 2009, 2011; Godel et al., 2012; Swart et al., 2012). Estimated at 20 giga base pairs (Gb) (Murray, 1998), sequen- cing and assembly of the genome of this gymnosperm species of the pine (Pinaceae) family present unique challenges. On the data generation end, those challenges include representation biases in whole-genome shotgun sequencing data, and difficulties in build- ing reduced representation resources to scale down the magni- tude of the problem. On the bioinformatics end, assembling massive sequencing datasets is extremely demanding on comput- ing cycles, memory usage, storage requirements, and for parallel programming implementations on communication traffic. We addressed the data representation challenges by preparing and sequencing multiple whole-genome shotgun libraries on the HiSeq 2000 and MiSeq sequencers from Illumina (San Diego, CA, USA). Compared with localized sequencing protocols, such as building and sequencing fosmid libraries, or the recent approach of isolating $10 kb DNA strands to generate indexed sequencing fragments in high throughput (Moleculo, San Diego, CA, USA), a shotgun only sequencing approach rapidly provides sequence data effectively covering the target genome at a cost that can be an order of magnitude less. The difference in cost is especially substantial when sequencing a large genome. In this work, we demonstrate that shotgun sequence assembly at this scale remains viable and produces valuable results. To *To whom correspondence should be addressed. ß The Author 2013. Published by Oxford University Press. at University of British Columbia on September 6, 2013 http://bioinformatics.oxfordjournals.org/ Downloaded from
  4. Sequencing Data • 65-fold coverage with HiSeq • 4-fold coverage

    with MiSeq 4 150 bp 150 bp 250 bp 11x 150 bp 150 bp 500 bp 54x 100 bp 100 bp 6 kb 8 kb 12 kb HiSeq 2000 300 bp 300 bp 500 bp 3x 500 bp 500 bp 500 bp 1x MiSeq
  5. 500-bp MiSeq reads Courtesy of Robin Coope @robincoope 5 Cartridge

    splitter MiSeq-XL cartridge base MiSeq-XL reagent tray & lid Screws for reagent tray lid Splash guard
  6. Merge overlapping reads FastQC plot of base quality Courtesy of

    Tony Raymond @tgjraymond 6
  7. Genome Assembly of White Spruce • Assembled using ABySS •

    Unitigs 1,560 cores and 5,460 GB of RAM for two days • Contigs 288 cores and 73 GB of RAM for four days • Scaffolds 36 cores and 62 GB of RAM for four days 7 White Spruce PG29 Published Latest ABySS version 1.3.5 1.3.7 Number of contigs (≥500 bp) 4.9 M 4.2 M N50 20.4 kbp 34.5 kbp Largest scaffold 1.05 Mbp 1.45 Mbp Assembled genome size 20.8 Gbp 20.8 Gbp
  8. Organellar Sequence in the Genome Assembly Courtesy of Tony Raymond

    @tgjraymond 8 ~6 Mbp
  9. Plastid Genome Photo credit Kristian Peters

  10. Plastid Genome Sequence • 4.7 million MiSeq read pairs of

    300 bp • Merged the overlapping paired reads • 3.0 million merged reads of 492 bp median • Assembled these reads using ABySS • Separated six plastidial sequences by
 length and depth of coverage 10
  11. The plastid genome Six scaffolds with depth of coverage >70x

    and length >5 kbp reconstruct the plastid 11 Six plastid sequences
  12. Plastid Genome Assembly • 125 kbp in six scaffolds with

    a 70 kbp N50 • Scaffold using 230 M mate-pair HiSeq read pairs • One circular scaffold of 125 kbp • 21 thousand reads (1/140 or 0.7%)
 map to the assembled plastid • 80-fold coverage of the plastid 12
  13. Plastid Genome Comparison • Aligned the white spruce plastid
 to

    the Norway spruce plastid • 99.2% identity and 98.8% coverage
 of the Norway spruce plastid • All 117 annotated genes are covered • 114 full length and 3 partial 13
  14. Mitochondrial Genome Illustration courtesy of Gary Carlson
 http://gcarlson.com/

  15. Mitochondrial Genome Sequence • 133 million HiSeq read pairs of

    150 bp • Filled the gap between the paired-end reads using a Bloom filter de Bruijn Graph (ABySS-connectpairs) • 1.4 million merged reads of 465 bp median • Assembled these reads using ABySS • 377 thousand merged reads (1/350 or 0.3%)
 map to the assembled mitochondrion • 30-fold coverage of the mitochondrion 15
  16. Mitochondrial Genome Assembly • Assembled one lane of HiSeq data

    using ABySS • 8.4 Mbp in 1001 scaffolds larger than 2 kbp with a 29 kbp N50 • Separated putative mitochondrial sequence by
 length, depth of coverage and GC content • 6.0 Mbp in 223 scaffolds larger than 2 kbp with a 39 kbp N50 • Scaffold using 230 M mate-pair HiSeq read pairs • 6.0 Mbp in 78 scaffolds larger than 2 kbp with a 157 kbp N50 • The largest scaffold is 519 kbp 16
  17. k-mer coverage vs GC content 17 Putative" mitochondrion

  18. Classifying the sequences using k-means clustering 18

  19. Mitochondrial Genome Comparison • The white spruce putative mitochondrial sequence

    is
 6.0 Mbp in 78 scaffolds larger than 2 kbp with a 157 kbp N50 • The Norway spruce putative mitochondrial sequence is
 5.5 Mbp in 294 scaffolds larger than 4 kbp with a 28 kbp N50 • 3.3 Mbp of these two assemblies align to each other with BWA • 98.3% identity and 59.6% coverage of the Norway spruce putative mitochondrial sequence 19
  20. Summary of Results • One lane of MiSeq data assembles

    the
 124 kbp plastid genome of white spruce • One lane of HiSeq data assembles the estimated
 6 Mbp mitochondrion genome of white spruce • Aligned to the complete plastid genome (NC_021456) and putative mitochondrial sequences of Norway spruce 20 Alignment Identity! Coverage Plastid 99.2% 98.8% Mitochondrion 98.3% 59.6%
  21. Further Work • Improve both assemblies by scaffolding
 and closing

    gaps • Annotate the genes of the plastid and mitochondrion • Determine whether the putative mitochondrial sequences are in fact mitochondrial
 (BLAST, circular scaffolds) • Investigate how the mitochondrial genome grew
 to such a large size 21
  22. Assembling the genome sequences of the plastid and mitochondrion of

    white spruce PAG 2014 Bioinformatics Workshop Shaun Jackman @sjackman 2014-01-14 22 Shaun D Jackman1, Anthony Raymond1, Ben Vandervalk1, Hamid Mohamadi1, René Warren1, Stephen Pleasance1,
 Robin Coope1, Macaire MS Yuen2, Christopher Keeling2, Carol Ritland2, Jean Bousquet3, Alvin Yanchuk4,
 Kermit Ritland2, John MacKay3, Steven JM Jones1, Jörg C Bohlmann2 and İnanç Birol1 (1) BC Cancer Agency, Genome Sciences Centre, Vancouver, BC, Canada, (2) University of British Columbia, Vancouver, BC, Canada,
 (3) Univesité Laval, Quebec, QC, Canada, (4) British Columbia Ministry of Forests, Victoria, BC, Canada Photo credit: Joseph O'Brien, USDA Forest Service, bugwood.org
  23. Fin

  24. Population Structure - Skimikin • Initial structure analyses • 5k

    random SNPs in HWE – 100k burn in – 200k MCMC generations – 1-15 genetic components (K), and K= 3 1/7/14
  25. Genome Sequencing of White Spruce PG29 25 Read Format Read

    Length (bp) Sequencing Platform Fragment Length (bp) # Libraries # Reads (M) Fold Coverage PET 150 HiSeq 2000 250 2 1,520 11.4 PET 150 HiSeq 2000 500 19 7,000 52.5 PET 300 MiSeq 500 4 170 2.6 PET 500 MiSeq 500 1 46 1.2 MPET 100 HiSeq 2000 6,000 1 268 7% MPET 100 HiSeq 2000 8,000 1 248 15% MPET 100 HiSeq 2000 12,000 7 34 60%
  26. Align the white spruce plastid to the Norway spruce plastid

    99.2% identity and 98.8% coverage 26
  27. Connecting Paired-end Reads 27 2x250 2x150 2x300 400 bp 500

    bp 600 bp Exists? Bloom Filter Courtesy of İnanç Birol
  28. Classifying using principle component analysis 28