Invalid format for mpileup use with VarScan

[Lien]

Dear all,

I'm trying to call variants on RNA-seq data.
I aligned the paired-end reads with Bowtie/Tophat and then generated a mpileup file with Samtools version 0.1.16 using the following command:
samtools mpileup -q 1 -Q 13 -f human_g1k_v37.fasta test.sorted.bam > test.sorted.mpileup

This mpileup file looks ok. Then I try to run VarScan version 2.3.2, with the following code:
java -jar VarScan.v2.3.2.jar mpileup2snp /test.sorted.mpileup --min-var-freq 0.08 --p-value 0.01 > /test.sorted.mpileup.varscan

Only SNPs will be reported
Min coverage: 8
Min reads2: 2
Min var freq: 0.08
Min avg qual: 15
P-value thresh: 0.01
Reading input from /test.sorted.mpileup

Initially, everything seems fine, but then VarScan throws me this error:

Error: Invalid format for pileup at line 419813325
22 31372123 A 1018 ...,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,......,.....,,,,,,,,,,,,,,............,,,,,,,,,,,,,,.....................,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.......................,,,,,,,,,,.........t,,..........................................,,,,,,,,,,,,,,,,,,,,,,,,,,,..........,,,,,,,,,,,,,,,,,,,,,,..,,,,,,,,,.,,,,.................,,...........................,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,..,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,....,,,,,,,,,,,...,,..,......................................................,,,........,,,,.....................................,,,,,,,............,,................................................,.........,.....,..............,.,,,,,........,.,,,,,,,..,,,,,,.,,,,,,,,.,,..,,,,,,,,,,,,,,,,,,,,,,,,,,,,.......,,,,,,,,,,,,,,,,...........,,,,.,,,,,,,,,,,,,,,,,,,,,,,,,......,,,,,,,..,,,,..,,,,,,,,,,,,,,,,,.............,,,,,.,,,,,,,,.....,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.GHJHHIJH@JIEECJI39<IG:9JI<DCDCD?JJJJIFJFGJ1JJIIHC)3DAHCC9G)?JJEEJHJBJ@:JIIHIJJGFAIIJIH@IDI@H?IIIJJIGEJJGJH1IHGJEJC?@EIIIJHJ@JIJJJFEJJHIIJFJEGJJFGHJGJHJIIIJHCGJJ?GJHJ?F<ACJIGEEFJBD9DACCC?FJHJJCIJEJJIGJJJIJJG@EJAJCJHEFJID?DCDC?#CDCCDC?DGJCDGIEFEHFBJH?GJIJJGGIC?<JD?IG@JAIFIIDIJHCD<CAIIGJJJJDJ;JEJIIIJGIEJEDHJ;GJJJIJGJIGG>@IJJIJCJJJGGJJC?HAHGFHADGCGHH?3<?DDH<<DGCFFDFDF?FDF#C???D@D#FFDDCCD?(3CDC<CCCCCC<D@+#DBDC#DDDA?C#9C?D@=CD@=9

The generated varscan-file up till that line seems fine. However, I don't really know how to work around this corrupt line. Would it be easiest if I just removed that line? And how can I do this on the mpileup file? Or are there other options?

Many thanks,
Lien

[dkoboldt]

It's strange to suddenly encounter an invalid-format line in SAMtools mpileup output. If I take it *exactly* as you pasted it, there's no delimiter (tab) between the last base call in column 5 and the first base quality value ("G"). However, if I put a tab between those, VarScan read the line just fine.

In terms of immediate action, you could simply remove the line (99.9% of reads show no variant anyway) with grep or vi or another command-line tool. You might also want to send me lines 419813320-419813330 of your pileup file and I'll take a look.

[Lien]

Hi Dan,

I also don't know how this strange line is formed. I performed the exact same commands on similar files, and they seem to work fine.
I just removed this line, so hopefully everything will work out now. I just wasn't sure if I could just delete this file without consequence.

Thanks for your help,
Lien

[vyellapa]

I have a similar error and am curious if the reason for this is found? Im running it in a pipeline and it would be easier if I could do something as a remedial step without having to check for the error messages.

Thank you,
Teja

Error: Invalid format for pileup at line 71
1       10277   C       0

[dkoboldt]

Vyellapa, can you send me the first 75 lines of your pileup file? Send it to dkoboldt (at) genome [dot] wustl [dot] edu

[dkoboldt]

Hello all,

We have just released VarScan v2.3.5 which should correct the invalid mpileup warning:

https://sourceforge.net/projects/varscan/files/

[wdemos]

v2.3.5 similar format error

I am also trying to call snps and indels using VarScan. I have the latest release (v2.3.5) installed so I can output to vcf format. I am using the following command and getting the invalid format error:

-bash-3.2$ java -jar VarScan.v2.3.5.jar mpileup2cns /research/sample.mpileup -min-coverage 8 --min-reads2 2 --min-var-freq 0.01 --min-avg-qual 15 --p-value 0.01 --strand-filter 0 --output-vcf 1 --variants 0 > /research/sample.vcf
Only variants will be reported
Min coverage: 8
Min reads2: 2
Min var freq: 0.01
Min avg qual: 15
P-value thresh: 0.01
Reading input from /research/sample.mpileup
Error: Invalid format for pileup at line 1
ï¿ï¿½BC��<�BCFYc1c2c3c4c5c6c7c8c9c10c11c12c13c14c15c16c17c18c19c20c21c22cXcYcMt5/research/Wdemos_work/sample_reordered_sorted.bam%##samtoolsVersion=0.1.17 (r973:277)

Can anyone please lend me a hand to figure out why it isn't working please? Also, I do not understand why the sorted. bam file in another directory is being referred to in the error message.

This is how I generated my pileup file:
samtools mpileup -q 1 -C50 -DSuf /ref/human_v37.fa /research/sample_reordered_sorted.bam > /research/sample.pileup

thanks

[wdemos]

the -u option was causing an issue. I was running it in a wrapper and not technically piping it in to VarScan

[dkoboldt]

Thanks for letting me know!

[hugorody]

short solution

Try to use this command before you call variants:

sed -n '/\t0\t/!p' file.mpileup > file.mpileup2

use the file.mpileup2 to call variants.

[bioliyezhang]

I have a similar error and am curious if the reason for this is found? Im running it in a pipeline and it would be easier if I could do something as a remedial step without having to check for the error messages.

Thank you,
Teja

Error: Invalid format for pileup at line 71
1       10277   C       0

Hi, Teja:

I wonder whether you solved the problem with mpileup, if so, would you mind giving me some suggestions on that? Thanks.

Best,
Liye

[vyellapa]

Looks like Dan Kabolt fixed it the next release of Varscan. If youre still having an error with the new release too, im not sure what else could help.