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Hi all,
A recent RNA-seq experiment of mine failed, and our core facility does not know why. Below is the disheartening email that I received this afternoon:
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We have finished sequencing your 15 cDNA samples. And during our data quality check step, we noticed your samples generated low number of reads, and also, the reads had low mapping ratio to reference genome. The average read numbers of the 15-sample project is 12.4 M, and the mean of mapped reads is 3.1 M. This is exceptionally abnormal, actually something we haven't seen before.
As for the trouble shooting, we found:
1. Regarding the library construction, we use an automatic prep station with the exactly same protocol to prepare all cDNA samples including your previous samples. The prep station has been used before and after your project, and all those results seemed normal.
2. Samples we sequenced along your sample, in the same lane or on same run, all performed very well in both read numbers and mapping. So the sequencing run was done with good quality.
3. Your samples' read sequences have a lot of GC repeats like following: NCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCAGGCGCGCGCAGGCGCGCGCCCGGGCAGCACGCGACCGCGCCGGCAGCACACCACGTGACGCA
GCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGAGCGCGGGAGACCACACGAGCGCACGCCAGCCACCCGCATGCCTCGCACGGCCGCCTCTGCTCGCACG
GCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCAGAGCGGACGCGCACGCCGCTGCACCCCAGGCACTGCCCACGTCCGTGTCCGCGCCGTCCGCTGAAAGA
It seems to us that your cDNA samples may contain some contamination. The dataset is in our downstream pipeline now. We can release them once it is done if you want to take a look.
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Has anyone seen this type of problem before? Any idea on what this mystery source of contamination could be?
For reference: I extracted whole RNA using the Arcturus PicoPure Isolation Kit, prepped cDNA using NuGen's Ovation RNA-seq System V2, and cleaned the samples with the Qiagen MinElute Reaction Cleanup Kit. Library prep was performed by the genomics core for sequencing on the Illumina HiSeq platform.
Right now I'm trying to figure out what to do with my remaining sample. Any advice would be welcome... these took me two years to collect and are beyond precious.
Thanks,
Amy
A recent RNA-seq experiment of mine failed, and our core facility does not know why. Below is the disheartening email that I received this afternoon:
-----------------
We have finished sequencing your 15 cDNA samples. And during our data quality check step, we noticed your samples generated low number of reads, and also, the reads had low mapping ratio to reference genome. The average read numbers of the 15-sample project is 12.4 M, and the mean of mapped reads is 3.1 M. This is exceptionally abnormal, actually something we haven't seen before.
As for the trouble shooting, we found:
1. Regarding the library construction, we use an automatic prep station with the exactly same protocol to prepare all cDNA samples including your previous samples. The prep station has been used before and after your project, and all those results seemed normal.
2. Samples we sequenced along your sample, in the same lane or on same run, all performed very well in both read numbers and mapping. So the sequencing run was done with good quality.
3. Your samples' read sequences have a lot of GC repeats like following: NCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCAGGCGCGCGCAGGCGCGCGCCCGGGCAGCACGCGACCGCGCCGGCAGCACACCACGTGACGCA
GCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGAGCGCGGGAGACCACACGAGCGCACGCCAGCCACCCGCATGCCTCGCACGGCCGCCTCTGCTCGCACG
GCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCAGAGCGGACGCGCACGCCGCTGCACCCCAGGCACTGCCCACGTCCGTGTCCGCGCCGTCCGCTGAAAGA
It seems to us that your cDNA samples may contain some contamination. The dataset is in our downstream pipeline now. We can release them once it is done if you want to take a look.
-------------------
Has anyone seen this type of problem before? Any idea on what this mystery source of contamination could be?
For reference: I extracted whole RNA using the Arcturus PicoPure Isolation Kit, prepped cDNA using NuGen's Ovation RNA-seq System V2, and cleaned the samples with the Qiagen MinElute Reaction Cleanup Kit. Library prep was performed by the genomics core for sequencing on the Illumina HiSeq platform.
Right now I'm trying to figure out what to do with my remaining sample. Any advice would be welcome... these took me two years to collect and are beyond precious.
Thanks,
Amy
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