getting-data

Obtaining data sounds simple but rarely is (in my experience) so I've created a repository to remind me of the processes as this is something I don't do very often since I usually create my own data.

NCBI-SRA archive

NCBI stores raw DNA sequences in .sra format, which need to be converted into fastq for whole genome processing pipelines. I followed the SRA Toolkit documentation but only managed to create files that were binary. Having no clue what this was about I went in search of someone who had also experienced this and found a GREAT article by the Edwards Lab at SDSU https://edwards.sdsu.edu/research/fastq-dump/ that explains why the NCBI documentation is so bad and how to get the files you need. Thank you https://github.com/linsalrob/EdwardsLab!

Required parameters

• Splitting reads

  --split-spot (splits spots into individual L and R reads, and puts them in the same singular file)

  --split-files (separates read into L and R ends, and puts FWD and REV reads in two separate files)

  --split-3 (separates read into L and R ends but if a L has not got a matching R, or vice versa, they will be put into a single file)

• Readids

  -I | --readids (appends read ID after spot ID as 'accession.spot.readid' on defline -> ID.1 = FWD read and ID.2 REV read)

  NB. --readids breaks BWA in downstream applications so this parameter may need to be omitted if BWA is in your pipeline. See Original format for more details.

• Technical sequences

  --skip-technical (dumps only biological reads)

• Clipping

  -W | --clip (applies L and R clips to remove SRA tags used in the whole genome amplification)

• Read filtering

  --read-filter (filters out reads recorded as N). Options are: pass | reject | criteria | redacted

• Original format

  -F | --origfmt (retains formatting of original definition line)

  SRA archive rewrites definition line in the seq. to include SRA ID and length if this parameter is omitted - this is a problem for BWA alignment as it doesn't recognize the new format and errors out >> i.e. [mem_sam_pe] paired reads have different names: "SRR849970.3.1", "SRR849970.3.2")

Optional parameters

• Sequence data formatting

  -B | --dumpbase (ensures that output is A, T, C and G instead of color space (used for SOLiD))

• Output to a specific directory

  -O | --outdir

• Compression

  --gzip

Example

cd ~/PATH/sratoolkit/bin

./fastq-dump < SRRxxxxxx > --outdir < ~/PATH > --gzip --skip-technical --readids --dumpbase --split-files --clip --origfmt --read-filter pass