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Raw

Phylogenomics of Acoelomorpha

  1. Fetch high quality RNAseq data from SRA.
  2. For each unique organism curate packages to be used.
  3. Include well annotated genomes.
  4. Insert raw data records into Agalma.
  5. Download data.
  6. Run Agalma phylogenomics pipeline.

Fetching SRA packages

I develop the python module fetch_sra.py to search and fetch packages from SRA Database. Using a defined set of criteria I (1) searched SRA packages, (2) filtered packages that matched these parameters. The commands are described in acoel_sra.py.

Search criteria:

  • Library Strategy: RNA-seq
  • Platform: Illumina or 454
  • Organism: Metazoa, but not Vertebrata or Insects
  • Modification data: After year 2000

Post-search filter criteria:

  • Library Layout: paired ends (with nreads=2)
  • Read Length: greater or equal than 70 bp

My initial search yield 3117 packages and 736 met our post-search filter requirements. The packages were written to paired_gte70bp_20140204.csv. These packages cover 91 unique organisms, shown in paired_gte70bp_20140204_unique.txt.

Package curation

Due to the large variety of sampled tissues, some manual curation of packages is needed to selected the most representative sequencing experiments.

TODO

  • Deal with previous Illumina ascii offset of 64.
  • Deal with reads that lack rrna (blast_rrna returns empty).
    • SRX096027, SRX096026.
  • Merge representative samples into single FASTQ pair.
    • cat runa_1.fastq.gz runb_1.fastq.gz > runa-runb_1.fastq.gz
    • cat runa_2.fastq.gz runb_2.fastq.gz > runa-runb_2.fastq.gz
  • Import previous Agalma assemblies from other databases.
  • Import other assemblies.
  • Import Genomes.

Test run

Test with Agalma's phylogenetic pipeline using 6 SRA packages.

species accession run million reads offset
Penaeus monodon SRX110652 SRR388222 29.6 33
Onchocerca volvulus ERX200391 ERR225731 39.2
Crassostrea virginica SRX118365 SRR404226 26.4
Hormogaster elisae SRX251927 SRR786599 30.0 33
Apostichopus japonicus SRX122622 SRR414930 26.9 33
Nanomia bijuga SRX288430 SRR871527 52.5 33
Haemonchus contortus SRX319246 SRR928056 29.6 64
Mizuhopecten yessoensis SRX220583 SRR653778 56.1 64 
Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Crustacea; Malacostraca; Eumalacostraca; Eucarida; Decapoda; Dendrobranchiata; Penaeoidea; Penaeidae; Penaeus; Penaeus monodon
Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Nematoda; Chromadorea; Spirurida; Filarioidea; Onchocercidae; Onchocerca; Onchocerca volvulus
Metazoa; Eumetazoa; Bilateria; Protostomia; Lophotrochozoa; Mollusca; Bivalvia; Pteriomorphia; Ostreoida; Ostreoidea; Ostreidae; Crassostrea; Crassostrea virginica
Metazoa; Eumetazoa; Bilateria; Protostomia; Lophotrochozoa; Annelida; Clitellata; Oligochaeta; Haplotaxida; Lumbricina; Hormogastridae; Hormogaster; Hormogaster elisae
Metazoa; Eumetazoa; Bilateria; Deuterostomia; Echinodermata; Eleutherozoa; Echinozoa; Holothuroidea; Aspidochirotacea; Aspidochirotida; Stichopodidae; Apostichopus; Apostichopus japonicus
Metazoa; Eumetazoa; Cnidaria; Hydrozoa; Siphonophora; Physonectae; Agalmatidae; Nanomia; Nanomia bijuga

Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Nematoda; Chromadorea; Rhabditida; Strongylida; Trichostrongyloidea; Haemonchidae; Haemonchinae; Haemonchus; Haemonchus contortus
Metazoa; Eumetazoa; Bilateria; Protostomia; Lophotrochozoa; Mollusca; Bivalvia; Pteriomorphia; Pectinoida; Pectinoidea; Pectinidae; Mizuhopecten; Mizuhopecten yessoensis

# Set resources.
export BIOLITE_RESOURCES="database=/sysdev/s9/bruno/acoel_phylo/biolite.sqlite,threads=24,memory=800G,outdir=/sysdev/s9/bruno/acoel_phylo/analyses"

cd /sysdev/s9/bruno/acoel_phylo/data/

sra import --clean --email organelas@gmail.com --gzip --metadata SRX110652
sra import --clean --email organelas@gmail.com --gzip --metadata ERX200391
sra import --clean --email organelas@gmail.com --gzip --metadata SRX118365
sra import --clean --email organelas@gmail.com --gzip --metadata SRX251927
sra import --clean --email organelas@gmail.com --gzip --metadata SRX122622
sra import --clean --email organelas@gmail.com --gzip --metadata SRX288430

catalog insert --id SRX110652 --paths SRR388222_1.fastq.gz SRR388222_2.fastq.gz
catalog insert --id ERX200391 --paths ERR225731_1.fastq.gz ERR225731_2.fastq.gz
catalog insert --id SRX118365 --paths SRR404226_1.fastq.gz SRR404226_2.fastq.gz
catalog insert --id SRX251927 --paths SRR786599_1.fastq.gz SRR786599_2.fastq.gz
catalog insert --id SRX122622 --paths SRR414930_1.fastq.gz SRR414930_2.fastq.gz
catalog insert --id SRX288430 --paths SRR871527_1.fastq.gz SRR871527_2.fastq.gz

cd /sysdev/s9/bruno/acoel_phylo/scratch/

agalma transcriptome --id SRX110652 > Pmon.out 2>&1 &
agalma transcriptome --id ERX200391 > Ovol.out 2>&1 &
agalma transcriptome --id SRX118365 > Cvir.out 2>&1 &
agalma transcriptome --id SRX251927 > Heli.out 2>&1 &
agalma transcriptome --id SRX122622 > Ajap.out 2>&1 &
agalma transcriptome --id SRX288430 > Nbij.out 2>&1 &

# Load.
agalma load --id SRX110652 --previous SRX110652
agalma load --id ERX200391 --previous ERX200391
agalma load --id SRX118365 --previous SRX118365
agalma load --id SRX251927 --previous SRX251927
agalma load --id SRX122622 --previous SRX122622
agalma load --id SRX288430 --previous SRX288430

# Homologize
agalma homologize 7 8 9 10 11 12 --restart homologize.chk --id PhyloTest > PhyloTest.out 2>&1 &

agalma multalign --id PhyloTest
agalma genetree --id PhyloTest
agalma treeprune --id PhyloTest
agalma multalign --id PhyloTest
agalma supermatrix --id PhyloTest
agalma genetree --id PhyloTest --raxml_flags="-o Nanomia_bijuga"

# Reports
agalma report --id PhyloTest --outdir reports/PhyloTest
agalma resource_report --id PhyloTest --outdir reports/PhyloTest
agalma phylogeny_report --id PhyloTest --outdir reports/PhyloTest

2nd test

agalma homologize 12 13 14 15 16 17 18 19 20 21 --id Phylo2