Ways to make a variant calling for RNA Seq (paired-end)

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1

Hello everyone! I’m a newer in bioinformatic and galaxy - sorry :mask:
I trying to use RNA STAR tool. I have full human transcriptom (RNAseq) in 8files (with R1 and R2 reads). So I have to get names of genes and their characteristics (pathogenic etc) in the end for clinicians.
I want to create vcf file for 1-snpsift variant type and 2-snpsift annotate SNPs from dbsnp. And after this I wanted download this vcf file (for ClinVar annotation etc).

I dont need the Trimmomatic so I was trying to do next:
Fastq–>RNA-STAR(hg38)–>Samtoolsmerge(for mapped.bam)—>FreeBayes

But FreeBayes show me the error:

  • { “code_desc”: “”, “desc”: “Fatal error: Exit code 101 ()”, “error_level”: 3, “exit_code”: 101, “type”: “exit_code” }
    Job API ID: bbd44e69cb8906b5339b2fad2d9562d3

Please explain me what I do wrong and which tools I need to use for my purpose?

2

Hi @Sofi

You should at least run FastQCand probably Fastq Info on your read data to make sure there is not a content/format problem.

These prior topics may also be helpful: Search results for 'FreeBayes order:likes' - Galaxy Community Help

Does the Dataset Details view contain any other informative messages? If not, check the prior topics for common help with this protocol, along with the tutorials below.

If you need more help, please post back a share link to the history containing this error. Please leave all inputs and outputs undeleted, along with the outputs from the QA checks. Sharing your History

Tutorials

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Thank you for answer. I’m sure for 1000% about high quality of my fastq-files. I made FastQC for files to demonstration it.
Here’s my history Galaxy
My problem is 106. FreeBayes.
I’ll be waiting for your commentaries :face_holding_back_tears:

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4

Hi @Sofi

It looks like you mapped against a different reference genome when running RNAStar (likely by accident).

Try rerunning those alignment jobs using the exact same reference database that is used for other steps → hg38. Then proceed with downstream steps using the newer BAM outputs.

5

Thank you, it really was a mistake. I used Samtools merge for my 4 .bam files after RNA STAR and file “samtools merge” is 2.3GB. But after FreeBayes file contains ~ 18k lines (7Mb)
Tell me please is it normal for RNAseq?
Can I use next tools for this file:
SnpSift Filter → SnpSift Annotate (+dbSNP_146_hg38.vcf)

6

Do you mean the VCF output? Yes, those are usually much smaller than BAMs.

Examples are included in the variant tutorials linked above.

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