I would like to check which DNA strands where my RNASeq have come from. I checked a couple of them by browsing UCSC:
R1 Read:
@HISEQ-WALDORF:249:C92LDANXX:8:1101:3161:1988
NTCGAGACTTCTTATAATTTGCATAATCCTCCAAAATGGAATCCACATTCTTCTTGGCAGGAAGATAAAAAAGCTGCTTCTGTCTGGTGATCAAGTCCCAGTCATCCACAAGCCAGGGTTTCAGC
+
#<<BBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF<FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF/FFFFFFFFFFFFFFFFFB
R1 UCSC Browse output indicates STRAND +:
ACTIONS QUERY SCORE START END QSIZE IDENTITY CHRO STRAND START END SPAN
---------------------------------------------------------------------------------------------------
browser details YourSeq 123 1 124 124 100.0% 9 + 90095138 90096554 1417
R2 Read:
@HISEQ-WALDORF:249:C92LDANXX:8:1101:3161:1988
CTACCGTTGAAAATGAAGAAACCTTCATGAACAGAGTTGAAGTTAAAGTGAAGATCCCTGAAGAGCTGAAACCCTGGCTTGTGGATGACTGGGACTTGATCACCAGACAGAAGCAGCTTTTTTAT
+
BBBBBFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF
R2 UCSC Browse output indicates STRAND +:
ACTIONS QUERY SCORE START END QSIZE IDENTITY CHRO STRAND START END SPAN
---------------------------------------------------------------------------------------------------
browser details YourSeq 123 1 125 125 99.2% 19 + 41525764 41525888 125
My question is whether this is a good way to check strands information. Is there other a quick and accurate way to check a whole fastq file? Desired output would be how many STRAND + and how many STRAND - from a fastq data.
samtools view -c -f 32 -F 256 foo.bam will give you one strand and samtools view -c -f 16 -F 256 foo.bam the other. Which is which will depend on how you did library prep.
Thanks @Devon Ryan! Could you have some explanation of the -f and -F options? And I got same numbers from my case:
samtools view -c -f 32 -F 256 C92LDANXX_s8_1_B01_0249_SL152940Aligned.out.bam
19770944
samtools view -c -f 16 -F 256 C92LDANXX_s8_1_B01_0249_SL152940Aligned.out.bam
19770944
What does it suggest?
Do you have paired-end reads? That's the most likely explanation for this.
Anyway, -F 256 means "ignore entries that are marked as secondary alignments". -f 32 means "only include alignments that are reverse complemented" and -f 16 means "only include alignments that are not reverse complemented".
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