samtools view -bT hg19.fa OR -bhs -q 30 -F1548 A.sam > A.bam, which one is better and what's the difference btw them?

Question

[Ming Lu]

I have seen two methods to transform .sam to .bam, wonder which one is better, "ref to genome sequence" OR "directly set -q and -F".

              samtools view -bT hg19.fa  A.sam > A.bam
              samtools view -bhs -q30 -F1548  A.sam > A.bam

which one is better?

Answer 1

[lakhujanivijay]

Step 1:

Check if the SAM file contains headers:

head A.sam

The first 10 lines on your terminal after typing "head test.sam" will be lines starting with the "@" symbol which is an indicator for a header line. If you don't see lines starting with the "@" sign, the header information is most likely missing.

The headers look like this:

@HD     VN:1.0  SO:coordinate
@SQ     SN:1    LN:248956422
@SQ     SN:2    LN:242193529
@SQ     SN:3    LN:198295559
@SQ     SN:4    LN:190214555
@SQ     SN:5    LN:181538259
@SQ     SN:6    LN:170805979
@SQ     SN:7    LN:159345973
@SQ     SN:8    LN:145138636
@SQ     SN:9    LN:138394717
@SQ     SN:10   LN:133797422
@SQ     SN:11   LN:135086622
@SQ     SN:12   LN:133275309

Step 2:

If the header is absent then run the command below, where hg19.fa is the reference fasta file used to map the reads:

samtools view -bT hg19.fa  A.sam > A.bam

If header is available:

samtools view -bS A.sam > A.bam

[Ming Lu]

Thanks a lot but still wonder when head available, why not samtools view -bhs -q30 -F1548 A.sam > A.bam

[lakhujanivijay]

From samtools help

-f INT   only include reads with all  of the FLAGs in INT present [0]
-F INT   only include reads with none of the FLAGS in INT present [0]
-q INT   only include reads with mapping quality >= INT [0]

From this link

-q 30 - include reads with mapping quality equal to or greater than 30

-F 154 - only include reads with none of the FLAGS in the image

I hope this helps !!

Answer 2

[ATpoint]

Both commands produce a proper bam file. The first command uses all entries in the sam. The second one filters out (-F 1548) unmapped reads and mates as well as reads that failed the quality control of the sequencer and marked duplicates. Also, -q 30 only takes reads qith a mapping quality above 30.