Sitka Spruce Mitochondrial Genome Assembly

2018-07-03

Shaun Jackman

Birol Bioinformatics Technology Lab
BC Cancer Genome Sciences Centre · Vancouver, Canada
@sjackman · github.com/sjackman · sjackman.ca

Previous Work

• Assembled and published the annotated sequences of
  complete white spruce plastid genome
  draft white spruce mitochondrial genome
  (Jackman et al. 2015)
• Assembled and published the annotated sequence of
  complete Sitka spruce plastid genome
  (Coombe et al. 2016)
• Currently working to assemble and annotate the
  draft Sitka spruce mitochondrial genome

Sitka Spruce Plastid

• One lane of Illumina 2x150 HiSeq of 10x GemCode
• One library rather than two:
  Illumina paired-end and mate-pair
• Assemble with ABySS
• Scaffold with ARCS and LINKS
• Close gaps with Sealer
• Polish with Pilon
• Annotate with Maker
• Manual annotation of difficult genes
• Complete plastid genome in one contig
• Perfect synteny to white spruce plastid

Sitka Spruce Mitochondrion

• 10x Genomics Chromium sequencing
• > 50x mitochondrial coverage in one lane
• 12 flowcells of Oxford Nanopore Sequencing
• 3x nuclear coverage
• 14x mitochondrial coverage

Assembly Strategy

• Assemble the Nanopore reads
• Polish the assembly using Chromium reads
• Check for misassemblies using Chromium reads

Nanopore reads

• Assemble Nanopore reads using Miniasm
• Polish the assembly using Racon
• Align reads to the draft assembly
  and separate putative mitochondrial reads
• Assemble mtDNA Nanopore reads using Canu

10x Genomics Chromium

• Assemble mtDNA Chromium reads using Unicycler
  guided by the Canu assembly
• Align Chromium reads to the assembly
• Check for misassemblies using Tigmint

Assembly

• 5.5 Mbp genome assembled in 5 contigs
• 3.54 Mbp, 881 kbp, 550 kbp, and 396 kbp
• One 168 kbp circular chromosome

Annotation

• Annotate genes using MAKER and Prokka
• 88 ORFs similar to 45 mitochondrial genes
  84 kbp or 1.5% of the genome
• 1,058 ORFS ≥ 300 bp
  409 kbp or 7% of the genome
• 8 rRNA genes (3 distinct)
• 25 tRNA genes (17 distinct)
• 10 Type II introns in 9 genes

Future Work

• Investigate genome structure
  and possible genomic isomers
• Validate genome structure with PCR
• Quantify expression using Nanopore RNA-seq

Shaun Jackman

Slides
https://sjackman.ca/psitchensismt-slides

Markdown source code
https://github.com/sjackman/
psitchensismt-slides