Aug 15 - Sept 9, 2019 Salmon-sea lice BS seq analysis

Sea lice bismark alignments:

• mox script here: https://gannet.fish.washington.edu/metacarcinus/mox_jobs/20190814_BmrkCalig.sh
• data here: https://gannet.fish.washington.edu/metacarcinus/Salmo_Calig/analyses/20190814_Calig/
• bismark summary report shows limited mehtylation as discussed on slack here. This isn’t that surprising as the global DNA methylation ELISA data showed Female 1 only 0.56% methylated and Female 2 3.38% methylated. We had tried WGBS with high-ish coverage to overcome the limited methylation
  • Sequencing depth:
    • aimed for 240M reads/sample
    • Female 1: 225.7
    • Female 2: ~163.7M
  • Methylated CpGs:
    • Female 1: ~2.15M
    • Female 2: ~1.33M
• Next steps:
  • still may be worth seeing what CpGs are present and if they are overlapping (if any have 5x coverage)
  • Maybe try ATAC-seq or different method to more sensitively detect the methylated DNA

Salmon bismark alignments:

Dealing with unplaced sequences in genome

• Aug. 14 alignments were to a genome that contained thousands of unplaced sequences:
  • figured this out from bamqc report plots
    • example:
    • 
  • see full preliminary bamqc reports
    • 16C_26psu_1_S13
    • 16C_26psu_1_S13
    • 16C_26psu_2_S14
    • 16C_26psu_3_S15
    • 16C_26psu_4_S16
• Talked with Steven and determined these unplaced sequences need to be removed and alignments redone
• Removing unplaced sequencings from genome:
  • jupyter notebook here: https://github.com/shellywanamaker/Salmon_sealice/blob/master/jupyter/Remove_unplaced_genome_seqs.ipynb
• copied new genome without unplaced sequences to mox
  • new genome fasta here: https://gannet.fish.washington.edu/metacarcinus/Salmo_Calig/GENOMES/v2/RefSeq/GCF_000233375.1_ICSASG_v2_genomic.fa
  • mox script to build bismark genome with new genome: https://gannet.fish.washington.edu/metacarcinus/mox_jobs/20190828_BmrkBld_Salmo1-29MT.sh
• mox script here: https://gannet.fish.washington.edu/metacarcinus/mox_jobs/20190828_BmrkBld_Salmo1-29MT.sh
  • new bismark genome here: https://gannet.fish.washington.edu/metacarcinus/Salmo_Calig/GENOMES/v2/RefSeq/chr1-29MT/

Alignments to bismark genome without unplaced sequences

• mox script here: https://gannet.fish.washington.edu/metacarcinus/mox_jobs/20190828_BmrkAlg_Salmo.sh
• data here: https://gannet.fish.washington.edu/metacarcinus/Salmo_Calig/analyses/20190828_Salmo/
• bismark summary report here: https://gannet.fish.washington.edu/metacarcinus/Salmo_Calig/analyses/20190828_Salmo/bismark_summary_report.html
• multisample BamQC report here:https://gannet.fish.washington.edu/metacarcinus/Salmo_Calig/analyses/20190828_Salmo/multisampleBamQcReport.html

DMR analysis

• ran preliminary DMR analysis with methylpy
  • mox script here:https://gannet.fish.washington.edu/metacarcinus/mox_jobs/20190907_DMRfindSalmo.sh
  • data here:https://gannet.fish.washington.edu/metacarcinus/Salmo_Calig/analyses/20190907_Salmo/
• Next steps:
  • no DMRs detected with current settings so need to rerun DMRfind with more liberal parameters