Follow Helio's pipeline

[yk-tanigawa]

objective

• Familiarize to work with nanopore data

  procedure

• Follow Helio's pipeline

  software installation

• poretools
• samtools
• LAST
• Picard

  data

• reference genome (transcriptome)
• nanopore data (of course)

[yk-tanigawa]

LAST installation completed

• They are in /share/PI/mrivas/bin/
• During the installation process, I got a trouble with gcc5, but gcc4.9.1 works fine 👍

[yk-tanigawa]

Corresponding ipython notebook

• https://github.com/rivas-lab/nanopore/blob/master/notes/20161018_mapping_and_counting.ipynb

[yk-tanigawa]

reference genome

• hg19 is downloaded and placed in ${PI_HOME}/data/hg19
• creating index files for LAST with lastdb command

[yk-tanigawa]

example of data

• I made a symbolic link to one of the cDNA file
• I converted the example file into FASTQ format with poretool fastq subcommand

[ytanigaw@sh-5-36 ~]$ ll data/
total 54
lrwxrwxrwx 1 ytanigaw mrivas   164 Oct 19 12:08 cDNA-sample.fast5 -> /home/ytanigaw/data/nanopore/20161006_minion_human_cDNA/DN0a27203a_SUNet_20161001_FNFAB30583_MN20225_sequencing_run_20161001_brcabl_80046_ch292_read263_strand.fast5
-rw-r--r-- 1 ytanigaw mrivas 15039 Oct 20 00:14 cDNA-sample.fq
lrwxrwxrwx 1 ytanigaw mrivas    26 Oct  6 10:51 nanopore -> /share/PI/mrivas/nanopore/
lrwxrwxrwx 1 ytanigaw mrivas    86 Oct 19 22:41 poretools-sample.fast5 -> /home/ytanigaw/software/poretools/test_data/2016_3_4_3507_1_ch120_read240_strand.fast5

[yk-tanigawa]

last alignment

• tried but got an error
  • https://github.com/rivas-lab/nanopore/blob/20161020lastal-err/scripts/20161019/lastal_cDNA_hg19.err

[yk-tanigawa]

• I still have the same problem, I can read the file from slum script (cat command works), but LAST says they can not read.
• I suspect there is an error in installation, so I am trying to see if we can reproduce the on scg4 cluster.
  • Luckily I have an access to LAST binary in Bustamante lab.

[yk-tanigawa]

• I found that the previous step with lastdb got an error
  • https://github.com/rivas-lab/nanopore/blob/20161020lastal-err/scripts/20161019/lastdb_hg19.err

[yk-tanigawa]

• After I re-run the lastdb step and lastal step, I obtained an alignment from last
• Next task would be counting mismatches from the results