RNA seq data preprocessing and cleaning - SEQanswers

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figo1019

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Join Date: Jun 2012

Posts: 32
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RNA seq data preprocessing and cleaning

07-17-2012, 12:45 AM

Hi All,

I am a total newbies in this field. I have to assemble RNA seq data. Before that I need to trim the sequences. I have got 100bp illumina paired end reads in two files. I also got the adaptors sequences P5 and P7.
5-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATC-(insert)-ACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTGXXXXXXTAGAGCATACGGCAGAAGACGAAC-3
I tried trim_galore... but still I can see these much of overrepresented sequences in the fastqc report.

ATGACACTCAAACAGGCATGCTCCACGGAATACCATGGAGCGCAAGGTGC 1155666 2.5956349017221085 No Hit
AATGACGCTCGAACAGGCATGCCCCTCGGAATACCAAGGGGCGCAATGTG 225179 0.5057538004361837 No Hit
AAGACACTCAAACAGGCATGCCTCTCGGAATACCAAGAGGCGCAAGGTGC 218636 0.4910581711090531 No Hit
GATCGTCGGACTGTAGAACTCTGAACGTGTAGATCTCGGTGGTCGCCGTATCATTAAAAA 119619 0.2686652123616139 Illumina RNA PCR Primer (100% over 50bp)
GATCGTCGGACTGTAGAACTCTGAACGTGTAGATCTCGGTGGTCGCCGTATCATTAAAAAA 111925 0.251384428005364 Illumina RNA PCR Primer (100% over 50bp)
AAATGACGCTCAAACAGGCATGCCCTTTGGAATACCAAAGGGCGCAATGT 104210 0.2340564774843778 No Hit
ACAAACCCTTGTGTCGAGGGCTGACTTTCAATAGATCGCAGCGAGGGAGC 71881 0.16144528987673504 No Hit
GATCGTCGGACTGTAGAACTCTGAACGTGTAGATCTCGGTGGTCGCCGTATCATTAAA 46463 0.10435626248303084 Illumina RNA PCR Primer (100% over 50bp)

I wanted to know how i can remove this overrepresented sequences from my data and do i need to remove it all ?
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Reply to Nine Things a Sample Prep Scientist Thinks About Before Sequencing

by GATTACAT

Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.
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by SEQadmin2

I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.

Here are nine questions we think about, in roughly the order they matter, before...
- Channel: Articles
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