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how to prepare a contaminant list for Bowtie2

I'd like to prepare a contaminant list to filter contaminant reads mapped to mitochondrial, ribosomal, actin RNA or phi X genomes28 bu Bowtie2.

Where can I download these sequeces for human and mouse?

Since it is the common contaminant, any available fasta files or bowtie index files for them?

Thanks~~

rna-seq
• 4,909 views • link
written by jolin0701-dy jolin0701-dy

Why do you want to filter those reads? Depending on the GTF file you use for read summarization reads that align to any/all of these would not be counted since you can exclude these features/meta-features from that GTF file.

• 2 views • link
written by GenoMax GenoMax

2 answers

I ran the index for phiX.

Then I want to run bowtie2 to exclude the sequences from fastq file.

./YZ1_S1_L001_R1_001.cuttrimmed.fastq is my input file.

./contam/phiX/phiX_index is the path to index

./nophiX.fastq is the output file exclude the phiX sequences.

$bowtie2 --un=./nophiX.fastq ./contam/phiX/phiX_index ./YZ1_S1_L001_R1_001.cuttrimmed.fastq

But the output file is 0kb and nothing is running.

Something wrong with the command?

Thanks….

• 1 views • link
written by jolin0701-dy jolin0701-dy

You may want to look at BBSplit from BBMap suite. This would be a much better tool for what you are trying to do and you can do all decontamination in one step.

• 1 views • link
written by GenoMax GenoMax

The order of parameters is important in case of most tuxedo suite programs (bowtie2 is one of them). Also lookup the correct parameters before using them.
Try

$ bowtie2 -x ./contam/phiX/phiX_index ./YZ1_S1_L001_R1_001.cuttrimmed.fastq --un ./nophiX.fastq
• 1 views • link
written by GenoMax GenoMax

I used it and shows

Error: Must specify at least one read input with -U/-1/-2 (ERR): bowtie2-align exited with value 1

Any thoughts?

Thanks~~

• 1 views • link
written by jolin0701-dy jolin0701-dy

I have corrected bowtie2 command line above. Try the new one now. Sorry about that.

• 1 views • link
written by GenoMax GenoMax

Thanks, it works.

Would you tell me how to run it in background and check the status of the running??

• 1 views • link
written by jolin0701-dy jolin0701-dy 
$ bowtie2 -x ./contam/phiX/phiX_index ./YZ1_S1_L001_R1_001.cuttrimmed.fastq --un ./nophiX.fastq &

Note the & at the end of the command that puts the job in the background. Do not close the terminal you start this from. If you look at a real time process monitor ($ top) you should be able to see the job running.

• 1 views • link
written by GenoMax GenoMax

I downloaded phiX genomes from NCBI and save it as phiX.fa

Then I ran bowtie2-build and got an error.

$ bowtie2-build phiX.fa phiX

Traceback (most recent call last):

File "/usr/local/bin/bowtie2-build", line 95, in <module> main()

File "/usr/local/bin/bowtie2-build", line 92, in main os.execv(build_bin_spec, argv)

OSError: [Errno 2] No such file or directory

Would someone tell me what's wrong with it?

Thanks~~

• 2 views • link
written by jolin0701-dy jolin0701-dy

Please use a different name for the index to avoid confusion with fasta file later on. See if the following works.

$ bowtie2-build ./phiX.fa phiX_indx

phiX_indx will become the basename of the index.

• 2 views • link
written by GenoMax GenoMax

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