Background

Conifer Genomics

• Conifer genomes are large, about twenty gigabases
• Before 2013 no conifer genomes had been published
• Then three in the period of one year!
  • Loblolly pine (Neale et al. 2014)
  • Norway spruce (Nystedt et al. 2013)
  • White spruce (Birol et al. 2013)

Genome Skimming

• Whole genome sequencing data contains both nuclear and organellar reads
• Each cell contains hundreds of mitochondria and plastids
• Reads of the organellar genomes are abundant
• Organellar sequences assemble well with a single lane
• Single-copy nuclear sequences are too low depth to assemble well

Classification

• Assembly is composed of
  • plastid sequence
  • mitochondrial sequence
  • nuclear repeat elements
• Identify putative organellar sequences by
  • homology to known organellar sequences
  • depth of coverage
  • length
  • GC content

Work so far

• 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

White Spruce Organelles

• Illumina paired-end
  • One 2x300 MiSeq lane for the plastid
  • One 2x150 HiSeq lane for the mitochondrion
• Illumina mate-pair for scaffolding
• Assemble with ABySS
  • Complete plastid genome in one contig
  • Draft mitochondrial genome in 36 scaffolds
• Close gaps with Sealer
• Polish with Pilon
• Annotate genes with Maker and Prokka
• Manual annotation of difficult genes
  • Three genes with short initial exons < 10 bp
  • One trans-spliced gene (rps12)

10x Genomics Chromium

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

Aim

Assemble the Sitka spruce mitochondrion into a single scaffold* using 10x Chromium data and annotate it.

* if it has a single chromosome

Method

• Align Sitka spruce reads to white spruce organelles with LongRanger
• Identify 10x barcodes that contain at least one mitochondrial molecule
  • Four properly-paired mitochondrial reads
• Assemble these mitochondrial barcodes with ABySS
• Scaffold with ARCS and LINKS
• Annotate genes with MAKER and Prokka

Classification

• Assembly is composed of
  • plastid sequence
  • mitochondrial sequence
  • nuclear repeat elements
• Identify putative organellar sequences by
  • homology to known organellar sequences
  • depth of coverage
  • length
  • GC content

Results

• Largest scaffold is 1.2 Mbp
• 50% of the 6 Mbp genome in 4 scaffolds > 460 kbp
• 75% of the genome in 13 scaffolds > 100 kbp
• 1/223 or 0.45% of reads are mitochondrial
• 115 ORFs with similarity to known mitochondrial genes
• 1,154 other ORFS ≥ 300 bp
• 9 Type II introns in 6 genes

fin

Shaun Jackman

Slideshttps://sjackman.ca/picea-sitchensis-organelles-slides

Markdown source codehttps://github.com/sjackman/ picea-sitchensis-organelles-slides

Links

ABySS · ARCS · Architect · Fragscaff · LINKS · LongRanger · MAKER · Pilon · Prokka · RNAweasel · Sealer · Supernova

Supplementary Slides

Future Work

• Identify mitochondrial barcodes using the draft sitka spruce assembly rather than white spruce assembly
• Fill gaps with Sealer and Kollector
• Polish with Pilon
• Identify introns genome-wide using RNAweasel
• Assemble with 10x Genomics Supernova

Scaffolding Tools for 10x

• ARCS with LINKS
• Architect intended for synthetic long reads
• Fragscaff intended for contiguity-preserving transposition