Hello all. I am trying to do a paired-end analysis. Ran the 2 fastq files with hisat2:
hisat2 -p 10 -x '..index_hg19/indexed' -1 R1_001.fastq.gz -2 R2_001.fastq.gz -S hisat2output.sam
Then :
samtools view -@ 10 -bS hisat2output.sam > hisat2.bam
samtools sort -@ 12 -n hisat2.bam > testSort.bam
samtools fixmate -m -@ 12 testSort.bam testSort_fixmate.bam
then i try to remove duplicates :
samtools markdup -r -@ 12 testSort_fixmate.bam > testSort_fixmate-markdup.bam
but returns problem :
ERROR: queryname sorted, must be sorted by coordinate.
WHEN i don't use -n option in samtools sort , samtools markdup returns problem:
ERROR: Coordinate sorted, require grouped/sorted by queryname.
I cant find where is the problem. Any help ?
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dimitrischat
edited answer:
You have to name-sort for samtools fixmate, and coordinate-sort for samtools markdup. Here is the correct order of operations, from samtools man file:
# The first sort can be omitted if the file is already name ordered
samtools sort -n -o namesort.bam example.bam
# Add ms and MC tags for markdup to use later
samtools fixmate -m namesort.bam fixmate.bam
# Markdup needs position order
samtools sort -o positionsort.bam fixmate.bam
# Finally mark duplicates
samtools markdup positionsort.bam markdup.bam
original answer
The problem is you are sorting by name (samtools sort -n), and you should sort by coordinate:
samtools sort -@ 12 hisat2.bam > testSort.bam
when i sort by coordinate it returns this problem :
[bam_mating_core] ERROR: Coordinate sorted, require grouped/sorted by queryname.
that worked. But could you tell me why my other pipeline was wrong? Do i need to do the one you suggested when its paired-end ?
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What does
samtools view -H testSort.bam | head -5show?Likely not the solution to your problem, but you can make these commands a lot shorter and avoid intermediate files:
By using pipes and directly going to
samtools sort:hisat2 -p 10 -x '..index_hg19/indexed' -1 R1_001.fastq.gz -2 R2_001.fastq.gz | samtools sort -n -o testSort.bamThis requires a reasonably recent version of samtools.
yes i do use them like you suggested. but now i am trying to figure out where the problem was. thats why i was running the commands 1 by 1
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