RNA-seq bacteria contamination
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5 weeks ago
sh • 0

Hello everyone!

Our lab now has a batch of single cell full length test data. After analysing it, I believe it has bacterial contamination. Do you have any more advice on this please? Thanks!

The cells are sequenced by MiSeq. and they are related to patient cells separated using Parsortix, stained with antibodies, single cells were picked with robot and then prepared libraries. In this workflow there are several steps, such as Parsortix separation that are not done in sterile conditions (not by a choice but due to the machine characteristics) and antibody staining. SIngle cell picking is performed in a protected enviroment of instrument chamber. Next, all steps during library preparation including clean up, perform only and exclusively under the PCR hood, using double filter tips, cleaning all the pipets etc.

I remove the adapters and perform genome mapping using STAR. I get a very low mapping rate 1.12% . Then I perform blast alignment for unmapped reads. And found they didn't align to human gene., most aligned to Cutibacterium acnes(taxid 1747) , Neisseriaceae bacterium(2014784), Staphylococcus epidermidis(1282). many blast alignment show that these sequences are 100% identical to the sequences of these bacteria.

RNA-seq • 220 views
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I get a very low mapping rate 1.12%

Hate to say this but if that result is real and accurate then cut your losses and start over. You may have lost the real sample somewhere along the way.

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