Hello everyone, I have lots of transcriptome data (RNA-Seq and full transcript single cell RNA-Seq) and I need to align them to genome using STAR. But I have storage some problems.
I will focus on a group of genes in downstream analyses. Is it possible to align the reads to specific parts of genomes (gene group) while using STAR or creating a custom reference from genome fasta that includes only specific regions and use it as reference?
Thank you in advance
That is possible but the question is it appropriate to do so.
If your data comes from entire genome/transcriptome then using a reduced representation reference always leads to the chance that STAR will try to align things to a location they may not have originated from.
please, don't. See Exome Sequencing: Masking The Non-Genic Sequences ?
So doing such a thing in the alignment step practically causes mis-aligned reads. I will try to align all reads to whole genome and extract the regions using a bed file. I believe this will not create false positives or mis-aligned reads (at least not as much as the previous scenario).
Thank you for your answers.
If the problem is the storage capacity, you can filter the bam file to those regions of interest, see this post: Extract Reads From A Bam File That Fall Within A Given Region.
I would not do that. With a few genes alone you are not going to do a meaningful analysis, for example normalization and DE needs a fair amount of genes to be robust. Even more so on single-cell level for QC purposes. If storage is limited then do it file by file, get bam, then the count matrix for a single sample. Delete bam, next one. Eventually concat the matrices into a single one. Or use salmon for everything which produces counts directly.
I wouldn't do that either:
Is it possible to align the reads to specific parts of genomes (gene group) while using STAR
I only proposed an alternative to the problem assuming that further analysis doesn't need information about other regions:
But I have storage some problems.
That might be a better solution (you should post it as an answer to the question, not to my answer):
If storage is limited then do it file by file, get bam, then the count matrix for a single sample. Delete bam, next one.
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