Hi, all,
I used STAR to align reads to zebrafish genome. I know STAR output soft-clipped reads. But when I Blat the read to genome and find that actually there are only two insertions and one mismatch in the read.
The reads is
@NB501962:91:HVTHLBGX5:4:11603:19437:19319
CTCTGATTCAGTCTACTCAGACTTGATTGACCCATCACAGAGCTCAAGTTTAAATTCTGAGTTTGTTGACCCTCC
+
AAAAAEEEEEEEEEEEEEEEAEEEEEEEE/EEEAEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEAE/EEEEE
The STAR command I used is:
STAR --runThreadN 48 --genomeDir ./annotation --outFilterMismatchNoverLmax 0.05 --readFilesIn Read.fastq --outFileNamePrefix test_ --outSAMtype BAM SortedByCoordinate --outSAMattributes Standard
The result is:
NB501962:91:HVTHLBGX5:4:11603:19437:19319 0 1 6002 255 54M21S * 0 0 CTCTGATTCAGTCTACTCAGACTTGATTGACCCATCACAGAGCTCAAGTTTAAATTCTGAGTTTGTTGACCCTCC AAAAAEEEEEEEEEEEEEEEAEEEEEEEE/EEEAEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEAE/EEEEE NH:i:1 HI:i:1 AS:i:53 nM:i:0
But when I check the genome browser and extract the sequence, the mapping should be like(underline is insertion, lowcase is mismatch):
read:CTCTGATTCAGTCTACTCAGACTTGATTGACCCATCACAGAGCTCAAGTTTAAATTCTGAGTTTGTTGAcCCTCC
gemo:CTCTGATTCAGTCTACTCAGACTTGATTGACCCATCACAGAGCTCAAGTTTAAA__CTGAGTTTGTTGAaCCTCC
I would expect it to be mapped as the whole read with insertion. But the 3' end sequence after insertion, STAR output it as unmapped (soft-clipped). If I use the EndtoEnd mode, this read can not be mapped to genome.
Can someone help me out with this? Or are there any parameters in STAR I can set to map this read properly?
Thank you in advance!
You might check the --outFilterMismatchNmax of your version. Using the length-depended MM-filter, you should that to 999 according to the "ENCODE" settings.
Hi Michael, As far as I know, --outFilterMismatchNmax is set to 10 by default. Besides, in my command line, I have used --outFilterMismatchNoverLmax 0.05, and my read length is 75, so the maximum mismatch should 75*0.05=3.75. In that read, there is one mismatch and two deletions. Do you know how STAR treats deletions? Even we assume deletions are counted as "mismatches". The score is still below the maximum, isn't it?
Hi, Pacome.pr,
Thank you for the suggestion, but as I meant in my post, I have tried the EndtoEnd mode, but this reads can't be mapped to genome.
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