Hi, I am doing environmental metagenomics for microbes. A lot of time we have t o do 16S rRNA amplicon sequencing and metagenomics. The reason is WGS metagenomics can't yeild a lot of rRNA gene reads. For example, if you have 1 million of metagenomic reads, you will get ~ 1000 reads of rRNA gene. The rule of thumb is 1 of 1000. I remember someone told me the current technique difficulties. He also says WGS tends to generate more reads coding for housekeeping genes than normal funnctional genes? Can anyone explain why is this? I know like HiSeq will do a bridge amplification like PCR. Is it because the universal primers/adapters using in the bridge amplification are not specific ?
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It has nothing to do with bridge amplification. WGS is whole-genome sequencing. The rRNA cluster is ~4500bp long, and a typical microbial genome is ~1000X as long. So only 1/1000 reads contain rRNA sequence. [Note: these are very rough size estimates and don't account for repeats of the rRNA genes, but it's a reasonable heuristic.] -
Ribosomal RNA contamination is fairly common for RNASeq. If you don't like the idea of amplification, have you tried RNASeq without the rRNA exclusion steps?Comment
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