如何运行这个 Bismark 亚硫酸氢盐测序程序？

Question

我对编码很陌生，所以我不确定如何处理这个问题。我想查看我们获得的一些数据并使用 Bismark 对它们进行排序。我已经使用 Trim Galore 来减少读取，现在我想将数据导入 Bismark。但是，我不确定如何解决这个问题。在文档中它说它需要 Perl 才能运行，所以我从 github 下载了 Perl 以及 Bismark zip 文件。我还下载了 bowtie2 zip 文件并将两个 zip 文件解压缩到同一个目录中。然后我打开 Perl 命令提示符并将目录设置为包含我提取的文件夹的目录。 我把这一行放在：

> \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.

在将目录更改为 Bismark 文件夹后，我也尝试过：

> 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

我尝试了其他一些方法，但总而言之，我对如何解决这个问题感到有些困惑。我现在下载的东西：

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

Bowtie2 压缩文件-https://github.com/BenLangmead/bowtie2

.fa 格式的基因组组装

我想以 fasta 格式分析的数据

任何见解都会有所帮助。

Answer 1

我认为Bismark 和bowtie2 仅原生支持 Linux 和 macOS。如果您想在 Windows 上使用 bismark，您可以尝试通过 *nix 仿真系统（如 Cygwin、MSYS2）安装它，或者直接使用 WSL。我在带有 Ubuntu 20.04 的 WSL 的 Windows 11 上对此进行了测试：

• 下载bowtie2-2.4.4-linux-x86_64.zip并解压到~/bowtie2/bowtie2-2.4.4-linux-x86_64文件夹。

• 下载Bismark-0.23.1.zip并解压到~/bismark/Bismark-0.23.1/

• 测试安装：

  $ 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>}
  [...]