Need help annotating..

[shyam_la]

I just used MuTect for calling mutations and it gave me 3800 mutations in a tab delimited text file.

The columns are given below, from the MuTect website (https://confluence.broadinstitute.or...GATools/MuTect)

What tool/tools can I use to annotate this data? And how should I go about it, since this output has what I believe are a lot of redundant columns?

You may also notice that output has quite a few columns in it. Here are some of the more prominent ones along with their definitions:

contig - the contig location of this candidate
position - the 1-based position of this candidate on the given contig
ref_allele - the reference allele for this candidate
alt_allele - the mutant (alternate) allele for this candidate
tumor_name - name of the tumor as given on the command line, or extracted from the BAM
normal_name - name of the normal as given on the command line, or extracted from the BAM
score - for future development
dbsnp_site - is this a dbsnp site as defined by the dbsnp bitmask supplied to the caller
covered - was the site powered to detect a mutation (80% power for a 0.3 allelic fraction mutation)
power - tumor_power * normal_power
tumor_power - given the tumor sequencing depth, what is the power to detect a mutation at 0.3 allelic fraction
normal_power - given the normal sequencing depth, what power did we have to detect (and reject) this as a germline variant
total_pairs - total tumor and normal read depth which come from paired reads
improper_pairs - number of reads which have abnormal pairing (orientation and distance)
map_Q0_reads - total number of mapping quality zero reads in the tumor and normal at this locus
init_t_lod - deprecated
t_lod_fstar - CORE STATISTIC: Log of (likelihood tumor event is real / likelihood event is sequencing error )
tumor_f - allelic fraction of this candidated based on read counts
contaminant_fraction - estimate of contamination fraction used (supplied or defaulted)
contaminant_lod - log likelihood of ( event is contamination / event is sequencing error )
t_ref_count - count of reference alleles in tumor
t_alt_count - count of alternate alleles in tumor
t_ref_sum - sum of quality scores of reference alleles in tumor
t_alt_sum - sum of quality scores of alternate alleles in tumor
t_ins_count - count of insertion events at this locus in tumor
t_del_count - count of deletion events at this locus in tumor
normal_best_gt - most likely genotype in the normal
init_n_lod - log likelihood of ( normal being reference / normal being altered )
n_ref_count - count of reference alleles in normal
n_alt_count - count of alternate alleles in normal
n_ref_sum - sum of quality scores of reference alleles in normal
n_alt_sum - sum of quality scores of alternate alleles in normal
judgement - final judgement of site KEEP or REJECT (not enough evidence or artifact)

[RDW]

With a few lines of Perl (or equivalent) you can convert the MuTect output into something that ANNOVAR can read - I think ANNOVAR needs explicit start and end positions for the variant, even if these are identical (as with a SNP), so you'll need to duplicate MuTect's single variant position column:

http://www.openbioinformatics.org/annovar/annovar_input.html

ANNOVAR only cares about the first 5 columns of data, but can (optionally) retain the other columns in its output, which can be useful.

[shyam_la]

With a few lines of Perl (or equivalent) you can convert the MuTect output into something that ANNOVAR can read - I think ANNOVAR needs explicit start and end positions for the variant, even if these are identical (as with a SNP), so you'll need to duplicate MuTect's single variant position column:

http://www.openbioinformatics.org/annovar/annovar_input.html

ANNOVAR only cares about the first 5 columns of data, but can (optionally) retain the other columns in its output, which can be useful.

I am a MD, not a Bioinformatician proper and writing a few lines of perl is beyond me. I would have given ANNOVAR a shot, if I had a .vcf file as output from MuTect..

In any case, I used SNPeff - it accepts text input now but support unfortunately is soon to be discontinued in later builds. I retained only the first 4 columns of MuTect output and it did a great job..

Thank you.

Q: Can IGV display annotated tab delimited text files graphically? Any other software recommendations for visualisation?

[krawitz]

Hi Shyam_la,
GeneTalk is designed for non Bioinformaticians analyzing human sequence variants. So this might be an option for you. Who gave you the data? There is a standard format for reporting variants in NGS resequencing projects. It is called variant call format, vcf. Usually your sequencing facility can provide your data in this format. The rest is automatically done in GeneTalk. you just upload the data and the annotation will be done automatically in the background. A tutorial video tutorial explains how to filter.
best,
peter

[RDW]

I am a MD, not a Bioinformatician proper and writing a few lines of perl is beyond me. I would have given ANNOVAR a shot, if I had a .vcf file as output from MuTect.

ANNOVAR can also accept text files - see my link. You just need an extra column that duplicates the variant position (since for SNPs, the start position is the same as the end). Any program that can manipulate tab-delimited text files can handle this, even Excel (just check that the first five columns don't get mangled!).

[JackieBadger]

BLAST2GO program

[shyam_la]

Hi Shyam_la,
GeneTalk is designed for non Bioinformaticians analyzing human sequence variants. So this might be an option for you. Who gave you the data? There is a standard format for reporting variants in NGS resequencing projects. It is called variant call format, vcf. Usually your sequencing facility can provide your data in this format. The rest is automatically done in GeneTalk. you just upload the data and the annotation will be done automatically in the background. A tutorial video tutorial explains how to filter.
best,
peter

Hi Peter,

Our sequencing facility provides only the raw reads. Im doing all the downstream analyses. I have perfected my pipeline upto annotation and my mutation caller MuTect provides only text output at the moment, as it is in beta stage (but provides excellent mutation calls in my opinion). vcf is not an option now..
Thank you anyway.

[shyam_la]

ANNOVAR can also accept text files - see my link. You just need an extra column that duplicates the variant position (since for SNPs, the start position is the same as the end). Any program that can manipulate tab-delimited text files can handle this, even Excel (just check that the first five columns don't get mangled!).

Oh, thank you! I had assumed for another source that annovar was not capable of that.. Will give it a try too now!!

[shyam_la]

BLAST2GO program

Just checked out their home page.. Sounds good! Will give it a shot right away..

[JackieBadger]

Just checked out their home page.. Sounds good! Will give it a shot right away..

If you want to know if these SNPs are non-synonymous (and you do not know the reading frame) you should use the tBLASTx against the nr database. Then once you have these results you can get GOannotations. You can also predict open reading frames using the OrfFinder program.