I am very new to coding so I'm not really sure how to approach this. I wanted to look at some data that we got and sequence them using Bismark. I already used Trim Galore to pare the reads, now I wanted to get the data into Bismark. However, I'm not exactly sure how to approach this. In the documentation it said that it required Perl to run so I downloaded Perl along with the Bismark zip file from github. I also downloaded the bowtie2 zip file and extracted both the zip files into the same directory. I then opened up the Perl command prompt and set the directory to one with my extracted folders. I put this line in:

> \bismark\bismark_genome_preparation --path_to_bowtie ^ 
    C:\Users\sevro\Documents\Lab_Code\bowtie2-master --verbose ^
    C:\Users\sevro\Documents\Lab_Code\genome
The system cannot find the path specified.

I also tried this after changing the directory to the Bismark folder:

> perl bismark
Failed to execute Bowtie 2 porperly (return code of 'bowtie2 --version' was 256). 
Please install Bowtie 2 or HISAT2 first and make sure it is in the PATH, 
or specify the path to the Bowtie 2 with --path_to_bowtie2 /path/to/bowtie2, 
or --path_to_hisat2 /path/to/hisat2

I tried a few other things but all in all I am a bit confused on how exactly to approach this. Things I have downloaded right now:

Bismark zip file- https://github.com/FelixKrueger/Bismark

Bowtie2 zip file- https://github.com/BenLangmead/bowtie2

A genome assembly in .fa format

The data that I want to analyze in fasta format

Any insight would be helpful.

CodePudding user response：

I think Bismark and bowtie2 only supports Linux and macOS natively. If you want to use bismark on Windows you can try install it via a *nix emulation systems like Cygwin, MSYS2, or simply use WSL. I tested this on Windows 11 with WSL with Ubuntu 20.04:

• Downloaded bowtie2-2.4.4-linux-x86_64.zip and extracted to ~/bowtie2/bowtie2-2.4.4-linux-x86_64 folder.

• Downloaded Bismark-0.23.1.zip and extracted to ~/bismark/Bismark-0.23.1/

• Tested installation:

  $ perl --version
  This is perl 5, version 30, subversion 0 (v5.30.0) built for x86_64-linux-gnu-thread-multi (with 50 registered patches, see perl -V for more detail)
  
  $ perl bismark --path_to_bowtie2 ../../bowtie2/bowtie2-2.4.4-linux-x86_64/Bowtie 2 seems to be working fine (tested command '../../bowtie2/bowtie2-2.4.4-linux-x86_64/bowtie2 --version' [2.4.4])
  Output format is BAM (default)
  Did not find Samtools on the system. Alignments will be compressed with GZIP instead (.sam.gz)
  Genome folder was not specified!
  
  DESCRIPTION
  
  The following is a brief description of command line options and arguments to control the Bismark
  bisulfite mapper and methylation caller. Bismark takes in FastA or FastQ files and aligns the
  reads to a specified bisulfite genome. Sequence reads are transformed into a bisulfite converted forward strand
  version (C->T conversion) or into a bisulfite treated reverse strand (G->A conversion of the forward strand).
  Each of these reads are then aligned to bisulfite treated forward strand index of a reference genome
  (C->T converted) and a bisulfite treated reverse strand index of the genome (G->A conversion of the
  forward strand, by doing this alignments will produce the same positions). These 4 instances of Bowtie 2 or HISAT2
  are run in parallel. The sequence file(s) are then read in again sequence by sequence to pull out the original
  sequence from the genome and determine if there were any protected C's present or not.
  
  The final output of Bismark is in BAM/SAM format by default, described in more detail below.
  
  
  USAGE: bismark [options] <genome_folder> {-1 <mates1> -2 <mates2> | <singles>}
  [...]