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Isomir Discovery Or Sequencing Contaminants?

Considering the RNA-seq contaminants list:

Illumina Small RNA RT Primer                    CAAGCAGAAGACGGCATACGA
Illumina 5p RNA Adapter                         GTTCAGAGTTCTACAGTCCGACGATC
Illumina RNA Adapter1                           TCGTATGCCGTCTTCTGCTTGT
Illumina Small RNA 3p Adapter 1                 ATCTCGTATGCCGTCTTCTGCTTG
Illumina Small RNA PCR Primer 1                 CAAGCAGAAGACGGCATACGA
Illumina Small RNA PCR Primer 2                 AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA
Illumina Small RNA Sequencing Primer            CGACAGGTTCAGAGTTCTACAGTCCGACGATC

I am trying to identify isomirs of certain miRNAs among the reads.

Reads(which scored at pattern matching):

TAGCTTATCAGACTGATG  TTGAC    ATC    
TAGCTTATCAGACTGATG  TTGA     ATC    
TAGCTTATCAGACTGATG  TTG      ATC   
TAGCTTATCAGACTGATG  TTGACA   ATC    
TAGCTTATCAGACTGATG  TTGACT   ATC    
TAGCTTATCAGACTGATG  TTGAA    ATC

Mature sequence: TAGCTTATCAGACTGATG

So, it can be observed that the nucleotides exceeding the pattern have some sort of resemblance with the end of the 3p adapter/small rna adapter(TTG) and the small rna 3p adapter/5p adapter(ATC).

For this dataset I have only removed the RNAAdapter1.

How can one distinguish between nucleotides that could lead to a possible isomir discovery and sequencing contaminants considering that the substrings are small (2-3 characters long)- in general?

rna illumina short next-gen sequencing
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updated by Jeremy Leipzig Jeremy Leipzig • written by Agatha • Agatha

1 answer

You will not encounter the 3' end of an adapter at the 3' end of a sequence. The ATC, though is probably, your untrimmed adapter. You should trim that using your trimmer/masker of choice.

You might be seeing differential cleavage. What is the hairpin sequence?

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written by Jeremy Leipzig Jeremy Leipzig

@Jeremy Leipzig- the hairpin looks like : TGTCGGGTAGCTTATCAGACTGATGTTGACTGTTGAATCTCATGGCAACACCAGTCGATGGGCTGTCTGACA

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written by Agatha • Agatha

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