Reordering Bam files based on chromosome number, when the chromosome format is chr1..etc

Question

[jparker4]

When trying to call variants I get the error message:

##### ERROR MESSAGE: Lexicographically sorted human genome sequence detected in reads. Please see https://software.broadinstitute.org/gatk/documentation/article?id=1328for more information. Error details: reads contigs = [chr1, chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17, chr18, chr19, chr2, chr20, chr21, chr22, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chrM, chrX, chrY]

Both my fasta file and my bams have the chromosomes sorted in this order. Therefore I need to re-sort my bams and possibly my fasta to be in numerical order: chr1,chr2 etc.

However, due to the chr in the chromosome name samtools sort does not sort the chromosomes into the desired order. Can anyone help with this sorting?

[GenoMax]

It would be useful to mention what program you are using to call variants.

[jparker4]

Apologies, that was misleading. It is part of a variant calling pipeline but the actual error occurs when trying to run SplitNCigarReads. The full command I am running is posted below the comment underneath this one.

Answer 1

[finswimmer]

Hello,

in the error message there is a link to the documentation about what is wrong and how you can solve this.

Version-specific alert for GATK 3.5

In version 3.5, we added some beefed-up VCF sequence dictionary validation. Unfortunately, as a side effect of the additional checks, some users have experienced an error that starts with "ERROR MESSAGE: Lexicographically sorted human genome sequence detected in variant." that is due to unintentional activation of a check that is not necessary. This will be fixed in the next release; in the meantime -U ALLOW_SEQ_DICT_INCOMPATIBILITY can be used (with caution) to override the check.

fin swimmer

(Aargh, third edit)

[jparker4]

Hi, thanks for the reply. Unfortunately the error is occurring when I run SplitNCigarReads, which requires the -U option ALLOW_N_CIGAR_READS. The tool will not let me pass two -U options in, so I'm a bit stuck.

Below is the error causing command:

java -Xmx10G -Djava.io.tmpdir=/fastdata/mbp15jdp/tmp -jar ~/Downloads/GenomeAnalysisTK-3.8-0-ge9d806836/GenomeAnalysisTK.jar                    -T SplitNCigarReads                     -R /shared/sudlab1/General/mirror/genomes/hisat/hg19.fa                    -I deduped.dir/GTEX-12WSH-Cerebellum.hisat.bam                     -o split.dir/GTEX-12WSH-Cerebellum.hisat.split.bam                     -rf ReassignOneMappingQuality                     -RMQF 255                     -RMQT 60                     -U ALLOW_N_CIGAR_READS

[finswimmer]

Hello,

ok than we have to resort your contig (that was my first solution before I saw the above information) even if I have no idea why GATK cares about the current order.

1. Sort your reference

$ samtools faidx reference.fa
$ cut -f1 reference.fa.fai|sort -k1V|parallel -k 'samtools faidx reference.fa {}' > ref_sorted.fa

2. Create a dictionary for the sorted ref

$ java -jar picard.jar CreateSequenceDictionary R=ref_sorted.fa O=ref_sorted.dict

3. Reorder bam file

$ java -jar picard.jar ReorderSam I=original.bam O=reordered.bam R=ref_sorted.fa CREATE_INDEX=TRUE

fin swimmer