I want to make pairwise comparisons of gene expression between tissue samples (same tissue, same species, different individuals) in 5 different treatments (with 2 biological and 2 technical -sequencing- replicates per treatment, using Illumina paired-end reads for de novo assembly). Before the differential expression analysis, I have to assemble a de-novo transcriptome.
Ideally, I'd like to have a good tradeoff between maximum recovery of splice variants and not too many computational chimeras.
Assuming unlimited computational resources, what would be the best strategy for pooling the samples in order to get a common set of transcripts for which to compare expression in different treatments. I thought of pooling all 20 samples for creating a single assembly that would contain the transcripts expressed in every condition and then I could map each sample to this assembly and subsequently compare. How much coverage is too much? (in terms of errors, chimeric sequences). My main concern is on how this will affect to the representation of isoforms from different treatments.
Is it more appropriate to make 5 different assemblies with 4 samples each and then collapse them with CD-HIT or a similar tool?
Thanks,
Carlos
Ideally, I'd like to have a good tradeoff between maximum recovery of splice variants and not too many computational chimeras.
Assuming unlimited computational resources, what would be the best strategy for pooling the samples in order to get a common set of transcripts for which to compare expression in different treatments. I thought of pooling all 20 samples for creating a single assembly that would contain the transcripts expressed in every condition and then I could map each sample to this assembly and subsequently compare. How much coverage is too much? (in terms of errors, chimeric sequences). My main concern is on how this will affect to the representation of isoforms from different treatments.
Is it more appropriate to make 5 different assemblies with 4 samples each and then collapse them with CD-HIT or a similar tool?
Thanks,
Carlos
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