I'm trying to streamline my metagenomics protocols for my lab. I have previously followed the 16S protocol (https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf) with minimal modification.
Can anyone explain what the purpose of the first cleanup step is? I understand that this removes primer dimers, adapters and other nasties, but is there any reason you can't just skip the first clean up, index your amplicon PCR products immediately after PCR, and then clean up post indexing, still removing undesirable products? In our case we would then proceed with library normalisation via Qubit, size selection via Blue Pippin or Pippin Prep, and sequencing.
Thanks!
Hi!
I think, you need to clean up a first PCR product before barcode attachment to get rid of primer leftovers, that will decrease the amount of chimeric sequences in your library. Also, some salts still may be present in PCR1 product.
If one/you don't care about the abundance/relative abundance or the accuracy and reliability of the data, sure one/you can do as you suggested.
But, single PCR will
1) generate a mess in the abundance profile of the reads (which is already not true in any PCR based method)
2) will produce reads with errors chimeric sequences and other PCR based errors
3) polymerase will produce errors in base incorporation
4) there could be inhibition in indexing step due to negative feedback mechanism of the nucleotide concentration
Timur Yergaliyev. Thanks for your answer.
I assume that the chances of chimeras forming are increased when skipping the cleanup as the template used in the indexing may contain leftover primers and incomplete copies of the target fragment? Just want to understand the mechanisms of why I do/don't do something.
Thanks!
Abhijeet Singh. Thanks for your answer.
I assume you meant single cleanup, not PCR. In relation to your answers:
1) Yes, I don't rely on relative read abundance at all as a measurement of biomass in my samples, but I do like to use it to very roughly compare major differences between the same taxon between samples. I understand there are still biases that affect that, but from a broad perspective it is nice to see what the results look like.
2) I assume this if for the same reason I have highlighted in my answer above?? This alone is a good enough reason for me.
3) So the first cleanup will strip any remaining polymerase from the previous PCR, reducing the rate of any errors in base incorporation in the index PCR?
4) I'm not sure I understand the mechanism behind this. Could you please explain in a little more detail?
Thanks very much for your thoughtful answers!
Owen Holland
Yes, but if we don't clean up the PCR1 reaction and proceed with the PCR2, we can still assume it as a single PCR reaction wise. But yes, single clean up is better to use in the context of your question.
1) If this is your purpose (to know what, and not how much of what), then I think you can use it, however, I am not sure if or whether it can cause any problem in sequencing process itself.
3) Theoretically, yes.
4) If the amplicon concentration is too much in the reaction, new amplicons will not be generated. In this case, new amplicons means indexed amplicons. At this case there are many possibilities.
PCR inhibition due to high DNA concentration is a common problem. Inhibition means after your final clean up you will not be having much of the indexed amplicons. Indexing will not change the fragment size and it will be difficult to see on the gel. And non indexed reads will not bind to the flow cell, thus bad sequencing run.
Abhijeet Singh
Thanks again for your response. This makes a lot of sense.
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