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I just finished the RNA-seq run on MiSeq machine using 2x250 v2 kit. The result is not very satisfied since there is only 74% cluster passing filter. Usually, the overclustering could cause low percentage of culster passing filter, but my cluster density is 986 K/mm2, which I do not think it is really over density (We ran Miseq sequencing several times with even higher cluster density, e.g. ~1,100 K/mm2, but we still could get pretty good % cluster PF, usually around 90%).
Here is my thoughts, which could possibly cause low % cluster PF:
1. Adapters on the both ends of my cDNA library.
First, I used Clontech SMARTer PCR kit to construct cDNA library from the whole animal. During the step for the 2nd strand of cDNA synthesis and cDNA amplification, 25nt primers were used to synthesize 2nd cDNA stran and amplify the cDNA to the proper concentration for the following sequencing application. Therefore, after the cDNA library was made, 25nt adapters (from the primers) were added to the both ends of every transcript/cDNA sequence. Then, I used NEBNext Ultra DNA Library Prep Kit for Illumina to make the library compatible to Miseq platform.
Illumina do the filter check for % cluster PF in earlier 25 cycles, as I'd read in some Illumina documentation. If it is the case, since the first 25 nt is adaptor, which will cause "low diversity" phenomenon, Machine probably "think" all my clusters are identical (Homogeneous library type) if only based on the first 25 nt. Eventally, this "homogeneous library" will cause low % cluster PF.
2. My 2x250 v2 kit was expired on Dec. 2014. Would the expired chemicals/kit cause low % cluster PF or, in other words, cause the poor quality of sequencing results?
Anyone has any idea about why I got low % cluster PF?
Thank you very much.
Here is my thoughts, which could possibly cause low % cluster PF:
1. Adapters on the both ends of my cDNA library.
First, I used Clontech SMARTer PCR kit to construct cDNA library from the whole animal. During the step for the 2nd strand of cDNA synthesis and cDNA amplification, 25nt primers were used to synthesize 2nd cDNA stran and amplify the cDNA to the proper concentration for the following sequencing application. Therefore, after the cDNA library was made, 25nt adapters (from the primers) were added to the both ends of every transcript/cDNA sequence. Then, I used NEBNext Ultra DNA Library Prep Kit for Illumina to make the library compatible to Miseq platform.
Illumina do the filter check for % cluster PF in earlier 25 cycles, as I'd read in some Illumina documentation. If it is the case, since the first 25 nt is adaptor, which will cause "low diversity" phenomenon, Machine probably "think" all my clusters are identical (Homogeneous library type) if only based on the first 25 nt. Eventally, this "homogeneous library" will cause low % cluster PF.
2. My 2x250 v2 kit was expired on Dec. 2014. Would the expired chemicals/kit cause low % cluster PF or, in other words, cause the poor quality of sequencing results?
Anyone has any idea about why I got low % cluster PF?
Thank you very much.
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