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A single read, say Read_X, is separately contained in two different fastq files, say A.fq and B.fq.
Both fastq files were aligned with BWA against three different indexes as follows:
First against a "human repeats" index. Unmapped reads were then aligned to a "human rRNA" index. The new set of unmapped reads were next aligned to a "human genomic (hg19)" index.
The bwa commands for all runs were:
Here is the puzzle: BWA treats Read_X differently in the two runs. For A.fq, the read passes through the first two indexes and is caught at (i.e., found to be aligned to) the third index. For B.fq, the read is caught at the first index itself.
What could be the possible reasons for this behavior? Ideally a given read should have the same fate if passed through the same indexes in the same order, or should it not? Below are the entries in the different SAM files.
A.fq alignments:
B.fq alignment:
Cheers,
BN
Both fastq files were aligned with BWA against three different indexes as follows:
First against a "human repeats" index. Unmapped reads were then aligned to a "human rRNA" index. The new set of unmapped reads were next aligned to a "human genomic (hg19)" index.
The bwa commands for all runs were:
Code:
bwa aln –l 0 –t 24 bwa samse –n 1
What could be the possible reasons for this behavior? Ideally a given read should have the same fate if passed through the same indexes in the same order, or should it not? Below are the entries in the different SAM files.
A.fq alignments:
Code:
1. "human repeats" SAM entry: Read_X 20 Repeat 32 0 43M * 0 0 <rev comp seq snipped> <rev qual snipped> XT:A:R NM:i:3 X0:i:0 X1:i:0 XM:i:3 XO:i:0 XG:i:0 MD:Z:27C2G4G7 XA:Z:Repeat, -308,43M,3; 2. "human rRNA" SAM entry: Read_X 4 * 32 0 43M * 0 0 <seq snipped> <qual snipped> 3. "human genomic (hg19)" SAM entry: Read_X 16 chr13 93985787 16 43M * 0 0 <rev comp seq snipped> <rev qual snipped> XT:A:U NM:i:1 X0:i:1 X1:i:5 XM:i:1 XO:i:0 XG:i:0 MD:Z:39C3
Code:
1. "human repeats" SAM entry: Read_X 16 Repeat 308 0 43M * 0 0 <rev comp seq snipped> <rev qual snipped> XT:A:R NM:i:3 X0:i:0 X1:i:0 XM:i:3 XO:i:0 XG:i:0 MD:Z:27C1C9C3 XA:Z:Repeat, -32,43M,3;
Cheers,
BN
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