Mutation analysis on RNAseq

Question

[Ron]

Hi all,

I am doing Mutation Analysis from RNA-seq data.I used RNAseqmut(https://github.com/davidliwei/rnaseqmut) to get the mutations.I want to annotate the mutations from the output generated using snpEFF. I am getting error while annotating the vcf file.

Command:

snpEff/SnpSift.jar annotate  /BED_GTF_References/dbsnp-V-146-All.vcf  {1}  > $TMPDIR/mutation_anno/{1/.}_dbsnp_annotated.vcf


**ERROR**
VcfFileIterator.parseVcfLine(115):  Fatal error reading file '/tmp/713110.1.standard.q/mutation_anno/top1000.txt' (line: 2):
chr1    13684   C   T   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   0   2   0   0   0   0   0   0   0   0   0   0   0   0   0   0
Exception in thread "main" java.lang.RuntimeException: java.lang.RuntimeException: WARNING: Unkown IUB code for SNP '0'
    at ca.mcgill.mcb.pcingola.fileIterator.VcfFileIterator.parseVcfLine(VcfFileIterator.java:116)
    at ca.mcgill.mcb.pcingola.fileIterator.VcfFileIterator.readNext(VcfFileIterator.java:167)
    at ca.mcgill.mcb.pcingola.fileIterator.VcfFileIterator.readNext(VcfFileIterator.java:56)
    at ca.mcgill.mcb.pcingola.fileIterator.FileIterator.hasNext(FileIterator.java:67)
    at ca.mcgill.mcb.pcingola.fileIterator.MarkerFileIterator.hasNext(MarkerFileIterator.java:64)
    at ca.mcgill.mcb.pcingola.snpSift.SnpSiftCmdAnnotate.annotate(SnpSiftCmdAnnotate.java:101)
    at ca.mcgill.mcb.pcingola.snpSift.SnpSiftCmdAnnotate.run(SnpSiftCmdAnnotate.java:290)
    at ca.mcgill.mcb.pcingola.snpSift.SnpSiftCmdAnnotate.run(SnpSiftCmdAnnotate.java:257)
    at ca.mcgill.mcb.pcingola.snpSift.SnpSift.run(SnpSift.java:354)
    at ca.mcgill.mcb.pcingola.snpSift.SnpSift.main(SnpSift.java:69)
Caused by: java.lang.RuntimeException: WARNING: Unkown IUB code for SNP '0'
    at ca.mcgill.mcb.pcingola.vcf.VcfEntry.parseAltSingle(VcfEntry.java:872)
    at ca.mcgill.mcb.pcingola.vcf.VcfEntry.parseAlts(VcfEntry.java:756)
    at ca.mcgill.mcb.pcingola.vcf.VcfEntry.parse(VcfEntry.java:721)
    at ca.mcgill.mcb.pcingola.vcf.VcfEntry.<init>(VcfEntry.java:102)
    at ca.mcgill.mcb.pcingola.fileIterator.VcfFileIterator.parseVcfLine(VcfFileIterator.java:113)

Does anyone would have any idea on this? Do I have to some filtration on the vcf file before doing annotation?

Thanks,

Ron

[swbarnes2]

That quoted line doesn't look like its vcf format.

Answer 1

[QVINTVS_FABIVS_MAXIMVS]

chr1 13684 C T 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Looks like a formatting error as swbarnes2 commented.

VCF files are formatted as such

CHROM   POS   ID   REF   ALT ....

Here's the documentation for VCFs https://samtools.github.io/hts-specs/VCFv4.2.pdf

You are missing the "ID" entry which might explain the error. Your file looks like it's formatted for PLINK or some other software tool. In the case of a REF=C and ALT=T you should only expect two alleles , encoded as 0 or 1. Your entry has 0 and 2 which is also not in correct VCF format. Additionally if your samples are diploid, the genotypes should be 0/0, 0/1, or 1/1 for the variant you supplied.

Alternatively, your file is almost in correct input format for ANNOVAR http://annovar.openbioinformatics.org/en/latest/

chr1 13684 13684 C T

works for ANNOVAR http://annovar.openbioinformatics.org/en/latest/user-guide/input/#annovar-input-file