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Overview

Using DNMTools to analyze whole-genome methylation sequencing data

DNMTools GitHub

DNMTools Read the Docs

Using SCG for storage and computing: Primer for SCG with information about nodes, modules, etc.

General setup with installing DNMTools and HTSLib

Using specific server

Using sessions to have separate jobs running simultaneously

  • tmux -- gives you a session and you can then switch between sessions
  • tmux control b, release everything, and then press d -- exit session
  • tmux ls -- see all the sessions; pwd and hostname to see that connected to server
  • tmux a -t15 (this connects to session 15, for example)

Setup using SCG modules

  1. Navigate to directory within vsebast/shared: cd /oak/stanford/scg/lab_vsebast/shared/wgms
  2. Want to load module for DNMTools using SCG
  3. module spider dnmtools
  4. module add dnmtools
    • advantage: the above method of installation is having issues, so this is a single step of loading a module
    • disadvantage: we cannot (re)install these pacakges as the most updated version (stuck with whatever SCG uses)
  5. Test that dnmtools is properly working by typing "dnmtools" (no quotes)
    • would not work in a separate session

Using DNMTools

Create an index using abismal: github & docs

  1. Indexing the genome
    • module load dnmtools
    • dnmtools abismalidx hg38.fa hg38.idx

Steps before mapping

  1. Decompress raw .fq.gz file
    • gzip -d <file.gz>
    • Faster to work with decompressed files, but they take more space
    • abismal can work with compressed files (in the process decompresses and then deletes)
  2. head <file.fq>
    • can see the content of the file
    • the first line is the location of the reads
    • the second line is the actual read: no C because there is conversion in methylation data (compared to seeing C in DNA data)
    • head <file.fq> -n2 will give 2 rows
  3. Removing adapters (trimming)
  4. Quality control (if you are not sure of quality of sequencing itself)

Mapping

  1. Mapping using abismal
    • General: $dnmtools abismal [OPTIONS] input.fq [input-r2.fq]
    • $dnmtools abismal -i $ind "$f"_1_val_1.fq.gz "$f"_2_val_2.fq.gz -t $p -v - ind is the genome index - "$f"_1_val_1.fq.gz "$f"_2_val_2.fq.gz are the two paired read files
    • f=d62_M38_CKDL230014191-1A_H5NYWDSX7_L2
      • Defining f in such a way that it is the same for the paired reads
    • module add trim-galore/0.6.7
    • trim_galore --paired -q 0 --length 0 "$f"_1.fq.gz "$f"_2.fq.gz
      • does quality control, removes adapters (i.e., all recommended steps)
      • output: trimmed version of file + _report.txt
      • slow process, but can make it 20-40x faster by doing multi process using "j" - trim_galore --paired -q 0 --length 0 "$f"_1.fq.gz "$f"_2.fq.gz -j 60