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Old 11-16-2014, 11:57 PM   #10
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,238
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Quote:
Originally Posted by bunce View Post
Hi, Sure - carry-over is always a possibility (we have been using the bleach wash protocol since february). I suspect (with no evidence) that people may 'blame' carry-over when it is on fact the lab workflow causing issues.
Illumina has acknowledged carry over issues: https://icom.illumina.com/MyIllumina...applications-m

If by “evidence” you mean how many people look for evidence of contamination source in their reads, I do not have an answer.

Quote:
I don't doubt that a 2-step PCR protocol works well - it will always generate a truck-load of library. The question is what portion or reads are chimeric or have tags that you have not used on that run? I would be interested if anyone (using 2-step PCR and re-using indexes), has quantified this?
Two step PCR if done properly does not generate truck load of amplicons. The aim should be number of cycles to generate enough yields for a QC and 3-4 sequencing runs which usually would be 50 ng>.

Chimeric reads actually would be more prevalent in one step PCR rather than two step, because two major reasons for Chimera formation during PCR are incomplete extension and strand invasion. The condition for both are favoured in one step PCR where the reagents are most likely to deplete and concentration of amplicons increase to a critical point favouring those reactions.

By using indexed primers for second step from a plate sealed with a plastic film and frozen, thawed and resealed multiple times, I have got 1 in 10,000 reads that had indices not used in the reactions. By using individual tubes containing an indexed primer and opening them one at a time such as recommended in Nextera protocol, no read was detected that had unused indexed primer. One obvious advantage using two step PCR with dual indexing (such as one described in Illumina 16S sequencing protocol, see link in #3) is that identifying and eliminating cross contamination post-sequencing (caused either by physical contamination during library prep or image analysis error with higher cluster densities) would be easy as chance of both primers being contaminated is reduced.

Quote:
Amplifying amplified DNA that then undergoes another amplification (cluster generation) can cause headaches downstream from chimera formation to contamination that builds over time. But, again, it does depend on the end use of the data.
Cluster generation should not encourage chimera formation because bridge amplification takes place in solid state and unlike in solution PCR, fragments are not free to interact with each other. Fragments that are in close proximity or hybridize to each other and may fuse and form a cluster, will not produce pure signal either because of presence of mix template amplification or multiple primer binding sites and will fail filter so they cannot contribute to chimera reads.

It is not clear to me what sort of contamination would built over time, if one follows standard molecular biology lab hygiene such as using disposable gloves, aerosol free tips and cleaning bench and pipette surfaces.
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