I am performing a 16S microbiome study, and have sent my samples to a lab for NGS using illumina MiSeq, until our own MiSeq here is up and running. I have received pictures of the preliminary microbiome PCR gel (25 cycles) and there is a strong band of the expected length (approx. 300bp) in all of the samples analyzed. A second PCR was then performed with sequencing adapters added, which displayed very faint bands on the gel, at a slightly higher molecular weight (due to the adapter integration).
I asked the sequencing company whether this was due to reduced number of cycles at the library prep stage and they confirmed that this was the case, with only 5 cycles to avoid PCR bias. Is this enough, or are more cycles than 5 usually used? The genomic DNA used for PCR template 16S amplification was extracted from the fish gut, and measured between 50-300ng/ul with Qubit analysis.
Hi Philip - Illumina protocols usually recommend 8-12 PCR cycles for the final library enrichment, but even 5 cycles can be enough. The sequencing facility usually requires libraries to have concentration of 2-10nM (but they use much less for the actual sequencing, e.g. 2-10pM). It is preferable to use the Bioanalyzer to check the quality and quantity of your final library. If you don't have access to the Bioanalyzer, but you have Qubit and have an idea of the average fragment size of your amplicons, you can calculate the molar concentration (e.g. see http://molbiol.edu.ru/eng/scripts/h01_07.html). The sequencing facility can/should do the quality check of your library before they proceed with sequencing anyway (Bioanalyzer and/or qPCR).
This is a late answer, but I have had success using 10 cycles. I have seen as low as 8 cycles. I have heard there are new Illumina primer barcodes that can be used in 1 step instead of 2 step PCR, which would save a lot of time and money. However, I haven't found them yet.
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