To find Single Nucleotide polymorphism
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5.2 years ago

Hi all, I am trying to find SNP in my dataset. I am trying to use SAMtools to get those. My input file is in fastq format. So, I am looking for help to follow to get the command line to use SAMtools. One important thing is in SAMtools they use bam files and I am looking to use fastq files. Regards.

RNA-Seq alignment next-gen snp • 1.4k views
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Step 1: you should align your clean fastq reads into your reference genome, such as hg38.fa, with Bowtie1/2/BWA and output sam files, then convert sam files into bam files using samtools. Step 2:you can use GATK or SAMTOOLS to call SNP from bam files.

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You forgot to ask a question.

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What have you tried?

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One important thing is in SAMtools they use bam files and I am looking to use fastq files

You can't wish for something and expect it to work. Since you realize that you must get the bam files from your fastq files, that is step 1 (hint: align your fastq files to a reference).

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I aligned the fastq file to the reference and got the mapped reads. Now I am using the fastq output to get the SNP. I am in wrong path?

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You want to use the BAM file which contains the alignment information. You don't want to extract the reads that mapped (which is what I assume you are alluding to?)

See the section on variant calling on this page that shows how to use Samtools (http://www.htslib.org/workflow/ ).

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I redid the alignment and now I am using variant calling. For variant calling, I have three reference file and I am not sure I can use all of them. I need your guidaince here.

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If you have three reference files (not sure what that exactly means, are they three versions of same reference or three independent ones?) then you can align your data independently to all three and then call variants on those alignments.

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Mapped reads are in sam or bam format, not fastq. Fastq is not the output of your alignment.

This looks like you are quite confused about what you are doing. Do you have someone in your institute (I suppose) who can guide you and put you in the right direction? An online Q&A forum is fine for (small) questions, but for an entire research strategy and a lot of support this might not be an optimal format.
However, if you can't find help you are definitely welcome here with your questions. It's just more convenient to talk directly to someone with experience.

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