how to prepare a contaminant list for Bowtie2
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5.2 years ago
jolin0701-dy ▴ 90

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 • 2.2k views
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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.

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5.2 years ago
jolin0701-dy ▴ 90

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….

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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.

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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
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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~~

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

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Thanks, it works.

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

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$ 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.

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5.2 years ago
jolin0701-dy ▴ 90

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~~

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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.

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