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Old 09-12-2017, 07:30 AM   #1
tomlodz
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Location: Poland

Join Date: Jul 2017
Posts: 10
Default Two Miseq runs - different quality

Hi everyone,

We try to do NGS on ancientDNA according to the protocol of this publication:
https://www.ncbi.nlm.nih.gov/pubmed/20516186

Library is prepared with custom adapters, single-indexed. The adapters used here resemble the most for the TruSeq LT kit. Also, custom primers are used for sequencing run.

We took two sequencing attempts on the V2 Nano 500 cycles Kit for MiSeq device. We did paired end reads 2x250. In both attempts, PhiX was added to the library.

In first run cluster density was far too low (100 mm2). This was probably due to the inhibition of the blunt-ending reaction in the first step of library preparation. What resulted in a low concentration of endogenous DNA. Beside this on my newbie eye overall run parameters are not perfect but acceptable.

In second run after real-time quantification concentration of the library was 5-6 x greater than previous. My calculations were correct because cluster density was now 500 mm2. Unfortunately, in this case the rest parameters of the run were horrible. Especially the first 10 cycles were very poor quality. This resulted in a lack of demultiplexing of the library because quality of Read 2 was far too low for analysis. I also notice that intensity was almost 10 x lower than the previous run but I donít know reason why ?

Both runs was prepared similar. Also good too know that ancientDNA sequencing is associated with the presence of unknown lengths of the DNA inserts in the library.

Can you please provide some advice? What cause such difference in quality of reads for this two RUNS. Most of the parameters are included in the attached pdf file. Thanks a lot!
Attached Files
File Type: pdf Miseq.pdf (1.62 MB, 49 views)
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