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Entering edit mode
5.4 years ago
Emilio Marmol ▴ 140

Hi everyone, I'm having some problems trying to figure out what sequence of adapter should I enter as input in cutadapt or trimmomatic to trim them from my fastqs.

I have a set of fastqs, each of them with a set of reads of 51 bp, comencing with an N and then a series of letters corresponding to the read. I have also the information about the index sequence in each fastq, after demultiplexing, and two sequences determining the primers used. For instance, this is the information about one fastqc I have:

@700470R:449:HVHH7BCXX:2:1101:1406:1948 1:N:0:GTGAAA
NGCAGCATTGTACAGGGCTATGAAGATCGGAAGAGCACACGTCTGAACTCC
+
#<DDDEHIIIIIIIIIIIIIIIHIIIIIIIIIIIIIIIIIIIIIIHIIIII
@700470R:449:HVHH7BCXX:2:1101:1814:1992 1:N:0:GTGAAA
NCCGGGTGCCGTAGGCTTAGATCGGAAGAGCACACGTCTGAACTCCAGTCA
+
#<DDDIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIH<DFGHIIIII
@700470R:449:HVHH7BCXX:2:1101:2184:1885 1:N:0:GTGAAA
NGGGGAGGTGGAGCAGATCGGAAGAGCACACGTCTGAACTCCAGTCACGTG
+
#<DDD<<CGHIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII


The index sequence is, as determined in the header, GTGAAA. I also have information about the SR primer, which is:

5 ́AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTGGGA3 ́

and the Index primer, which is:

5 ́CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT3 ́

Substituting the NNNNNN with the index sequence provided in the header of the corresponding fastq, I would obtain the barcoded adapter used for sequencing, if I'm not wrong.

So here is where I start getting lost. After doing fastqc analysis, I got a list with a bunch of sequences in the overrepresented sequences, corresponding to Illumina Multiplexing PCR primer, as if there were different adapters withing the whole fastq in the same file.

So, here is my question:

As you all see... quite lost I am...

RNA-Seq microRNAs trimming adapter sequencing • 2.4k views
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Entering edit mode

i'd just use trimmomatic because it has built in multi thread support and just chuck out all the highly over represented sequences and if the reads are too short as a result chuck them out too. then do some basic quality trimming as well & chucking out. you can just read the manual and play around, it is a well written tool.

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