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Old 02-18-2019, 02:58 AM   #1
Marjun Berbisá
Junior Member
Location: Faroe Islands

Join Date: Feb 2019
Posts: 1
Default are the samples clean enough for sequencing


Here are my questions:
I am collecting stool samples from patients and preparing v4 amplicon libraries for sequencing on the MiSeq using the goLay barcode strategy with primer set 515F/806R which expects a size around 380-400

1. How do you determine if the samples are clean enough for sequencing? I´m concerned about doing multiple rounds of Ampure bead clean-up and losing all my PCR product if it isn´t necessary. I have attached the Bioanalyzer traces for reference.
2. As a trial run, we are thinking about running 21 samples + positive and negative controls with 50% PhiX to be super conservative just to see that we get usable data. We plan on using the V2-300 cycle MiSeq kit and the new 3.1 software is getting installed this week. What is your advice on this? Do you have a step-by step instruction on how to set up the run using the Golay barcode method and these samples?
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