Hi all,
(If a similar question has already been posted, kindly direct me to the answer). We have just sequenced a bacterial transcriptome under different growth conditions and one of the problems we had was that out of 13million reads, around 12 million reads mapped to unannotated regions. If you blast the sequence, it is apparently rRNA. That left me with only 1 million reads that actually map to the genome, and I have used this to calculate gene expression values (RPKM) - this means that the coverage per gene is very low, and possibly we miss out on genes with low levels fo expression. The total RNA was enriched for mRNA using MicrobeExpress (Ambion) by the external sequencing facility. My question is whether anyone has faced a similar problem, and what can we do to improve mRNA enrichment?
Sorry for the long message and I look forward to your answers.
(If a similar question has already been posted, kindly direct me to the answer). We have just sequenced a bacterial transcriptome under different growth conditions and one of the problems we had was that out of 13million reads, around 12 million reads mapped to unannotated regions. If you blast the sequence, it is apparently rRNA. That left me with only 1 million reads that actually map to the genome, and I have used this to calculate gene expression values (RPKM) - this means that the coverage per gene is very low, and possibly we miss out on genes with low levels fo expression. The total RNA was enriched for mRNA using MicrobeExpress (Ambion) by the external sequencing facility. My question is whether anyone has faced a similar problem, and what can we do to improve mRNA enrichment?
Sorry for the long message and I look forward to your answers.
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