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I aligned RNA-Seq reads to the reference genome. However, reads that span splice junctions will not align. What is the best way to handle this? I've read that aligning to a reference transcriptome is not the answer. Any thoughts? Thanks!
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You might want to try TopHat/Bowtie.
Alternatively, you can download all mRNA sequences from UCSC and align against that.
Sam -
Thanks for the suggestions...I would like to stick with BWA....downloading only RNA sequences will cause novel genes to not align correctly. Any other thoughts?Comment
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Maybe SpliceMap?
You know, you could always try TopHat. It's not like sequencing data goes bad after so many alignments
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So I used tophat 2 to align the data. I used multiple cores but ran it on the default settings.
I have tumor and normal samples (no replicates, single end).
My accepted_hits.bam for the normal samples is 479MB while the unmapped.bam file is 3.4 GB. I was wondering what I might be able to do about this? I am re-aligning the unaligned reads in BWA, but I'm not sure if this is the best approach. Any thoughts? There should not be so few mapped reads for this sample. The tumor samples had an unmapped.bam file of size 796 MB and an accepted_hits.bam file of size 479 MB. Although this is better, it still seems low.
The normals should actually be better than the tumors due to sample quality.
Thanks in advance for your help!Comment
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Did you quality trim before running the alignment? If not and a lot of your reads deteriorated in quality toward the end, that could be the issue. You might also try blasting a couple of the unmapped reads just to ensure there's nothing weird going on.Comment
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In that same vein, did you do any adaptor trimming? That might be an issue if you haven't.
SamComment
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...06-18-2026, 07:11 AM -
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
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