Freebayes - error in SNP calling

Question

[krp0001]

Hello all,

I am facing a problem in FreeBayes, I am calling for variants in a haployid population. After I submit the job, the stdeerr file keeps increasing in size and it has error stating 'Opened BAM reader without index file, jumping is disabled.

HISEQ:115:C49JYACXX:4:1206:9623:100139 at scaffold1|size923514:1 is out of order!
HISEQ:56:D1UAHACXX:2:1113:3701:14592 at scaffold1|size923514:2 is out of order!
HISEQ:115:C49JYACXX:4:1210:15069:58013 at scaffold1|size923514:1 is out of order!

so on.., I am not sure how long it takes to get the final vcf file. But for it keeps running for more than 20 days and job gets aborted due to internal issues. The size of vcf file is too high 40gigs.

Can you please help with the issue, this is my command I am using:

freebayes \
  -0 \
  --use-best-n-alleles 4 \
  --ploidy 1 \
  -C 2 \
  --no-indels \
  --no-mnps \
  --no-complex \
  -f ..bam

As suggested by Erik Garrison I sorted and indexed the bam files, I still face the same problem. I am not sure whether to feed the index file or not and how to feed.

Any help highly appreciated thanks in advance.

Prakash

[Adrian Pelin]

Okay, try sorting and indexing your bam file, it's really important. See if the errors go away.

[krp0001]

Thank you Adrian, I did both sorting and indexing, below is the script that I haven been using

freebayes \
  -0 \
  --use-best-n-alleles 4 \
  --ploidy 1 \
  -C 2 \
  --no-indels \
  --no-mnps \
  --no-complex \
  -f ../../assm_1kb.fasta \
  -b 1.bam 2.bam ......bam > output.vcf

Thank you

Answer 1

[Adrian Pelin]

My pipeline was the following, map with bwa, generate bam, sort bam, then:

java -jar ~/programs/picard-tools-1.94/AddOrReplaceReadGroups.jar I=GDR-18.sort.bam O=GDR-18.sort.grp.bam LB=whatever PL=illumina PU=whatever SM=whatever VALIDATION_STRINGENCY=LENIENT
java -jar ~/programs/picard-tools-1.94/MarkDuplicates.jar INPUT=GDR-18.sort.grp.bam OUTPUT=GDR-18.sort.grp.md.bam METRICS_FILE=GDR18.duplicates REMOVE_DUPLICATES=TRUE ASSUME_SORTED=TRUE VALIDATION_STRINGENCY=LENIENT
freebayes --fasta-reference gdr18_k75-85_NHC_conc.fasta --ploidy 4 --pooled-continuous --min-coverage 10 -F 0.1 --vcf GDR-18.vcf GDR-18.sort.grp.md.bam