Hello,
I am very new to Illumina sequencing, so I apologize if this is a very basic question. I tried searching other threads and didn't see anything similar to my problem.
Anyway, in March I constructed a 3-plex RNA-seq library as a preliminary run, and got excellent results. I wanted to add more replicates to the study, so using the same kit, same protocol, and biological replicates of the same RNA samples (collected at the same time as the prelim samples and stored at -70), I constructed 6 more libraries for 2 more 3-plex lanes. The bioanalyzer plots (attached) indicated that the libraries contained the expected fragment sizes. Also, the sequencing facility quantified my libraries with qPCR, indicating that the the library fragments did indeed have ligated adaptors.
However, when the libraries were sequenced, the results were very poor. Namely, the intensity plot for the G base (also attached) showed that at most positions, there was no G signal. My contact at the sequencing facility is looking into the cause of this problem, but he was very surprised as well, given that the libraries passed QC. He suggested it may be due to rRNA contamination, but (at least according to my novice thought process) there should still be some G signal, even if rRNA is being sequenced.
Has anybody seen this, or does anybody have any insight into what could cause this problem?
Thanks in advance for your input.
nicholas
Teets_DNA1000 Agilent_8-10-11-1.pdf
Intensity graph.pdf
I am very new to Illumina sequencing, so I apologize if this is a very basic question. I tried searching other threads and didn't see anything similar to my problem.
Anyway, in March I constructed a 3-plex RNA-seq library as a preliminary run, and got excellent results. I wanted to add more replicates to the study, so using the same kit, same protocol, and biological replicates of the same RNA samples (collected at the same time as the prelim samples and stored at -70), I constructed 6 more libraries for 2 more 3-plex lanes. The bioanalyzer plots (attached) indicated that the libraries contained the expected fragment sizes. Also, the sequencing facility quantified my libraries with qPCR, indicating that the the library fragments did indeed have ligated adaptors.
However, when the libraries were sequenced, the results were very poor. Namely, the intensity plot for the G base (also attached) showed that at most positions, there was no G signal. My contact at the sequencing facility is looking into the cause of this problem, but he was very surprised as well, given that the libraries passed QC. He suggested it may be due to rRNA contamination, but (at least according to my novice thought process) there should still be some G signal, even if rRNA is being sequenced.
Has anybody seen this, or does anybody have any insight into what could cause this problem?
Thanks in advance for your input.
nicholas
Teets_DNA1000 Agilent_8-10-11-1.pdf
Intensity graph.pdf
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