Question regarding the proposed varscan command

[gilhornung]

Hi Christoffer,

More of a question than an issue. I'm trying to run the superFreq with the variant calling that you suggested in your manual. Here are the commands that I use (which essentially follow your command):

samtools mpileup -d 10000 -q 1 -Q 15 -A WS4-C.bam > WS4-C.pileup
java -jar VarScan.v2.3.9.jar  mpileup2cns WS4-C.pileup --variants \
   --min-coverage 7 --strand-filter 0 --p-value 0.01 --min-var-freq 0.01 > WS4-C.varscan

The output file seem to have a line for essentially every base in the genome (see head below). Does that make sense? Should VarScan filter out some of the "variants"?

Chrom   Position        Ref     Var     Cons:Cov:Reads1:Reads2:Freq:P-value     StrandFilter:R1+:R1-:R2+:R2-:pval       SamplesRef      SamplesHet      SamplesHom  SamplesNC        Cons:Cov:Reads1:Reads2:Freq:P-value
1       10032   N       A       A:7:0:7:100%:2.9138E-4  Pass:0:0:5:2:1E0        0       0       1       0       A:7:0:7:100%:2.9138E-4
1       10033   N       A       A:8:0:8:100%:7.77E-5    Pass:0:0:7:1:1E0        0       0       1       0       A:8:0:8:100%:7.77E-5
1       10034   N       C       C:9:0:9:100%:2.0568E-5  Pass:0:0:7:2:1E0        0       0       1       0       C:9:0:9:100%:2.0568E-5
1       10035   N       C       C:10:0:10:100%:5.4125E-6        Pass:0:0:8:2:1E0        0       0       1       0       C:10:0:10:100%:5.4125E-6
1       10036   N       C       C:11:0:11:100%:1.4176E-6        Pass:0:0:9:2:1E0        0       0       1       0       C:11:0:11:100%:1.4176E-6
1       10038   N       A       A:15:0:13:92.86%:9.6148E-8      Pass:0:0:12:1:1E0       0       0       1       0       A:15:0:13:92.86%:9.6148E-8
1       10039   N       A       A:14:0:14:93.33%:2.4927E-8      Pass:0:0:11:3:1E0       0       0       1       0       A:14:0:14:93.33%:2.4927E-8
1       10040   N       C       C:15:0:15:100%:6.4467E-9        Pass:0:0:11:4:1E0       0       0       1       0       C:15:0:15:100%:6.4467E-9
1       10041   N       C       C:16:0:16:100%:1.6637E-9        Pass:0:0:12:4:1E0       0       0       1       0       C:16:0:16:100%:1.6637E-9

[gilhornung]

Just realized that this may be because the --fasta-ref option of the pileup is missing. Maybe correct the manual?

Thank you

Gil

[ChristofferFlensburg]

yeah, you are right, that's due to not having a fasta in the samtools call. I updated that. I see I haven't touched the manual in a while otherwise.