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After I use the "-q 20" option in bwa aln I start getting errors down the road eg when i run ValidateSamFile in picard I get lots of "Mate negative strand flag does not match read negative strand flag of mate" and then "Sequence and quality are inconsistent" errors as well. I have run all the same commands without the -q option without these problems. I am very new to all this and not sure where I'm going wrong!
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A little late, but I am also running -q option and curious if you identified the problem. -
No I ended up just running it without trimming as I decided it wasn't really necessaryComment
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This is a bug in BWA. If you use -q it puts the trimmed nucleotide read in the output SAM file but the quality sequence is not trimmed at all and thus there is a difference in the length of the read and the length of the quality string. Either use some third party tool like trimgalore to trim your fastq reads before aligning and don't use BWA -q option at all OR use some script that takes SAM file and trims off the base quality string accordingly.
PS:This post may help- http://www.biostars.org/post/show/13...or-space-data/Comment
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Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM -
With the launch of new single-cell sequencing platforms in 2026, the field stands at an exciting inflection point. This article surveys the most impactful advances in the field and discusses how they’re reshaping research in cancer, immunology, and beyond.
Introduction
Single-cell sequencing technologies have undergone remarkable advances over the past decade, transitioning from low-throughput experimental approaches to highly scalable platforms capable of...05-22-2026, 06:42 AM
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