Question: Hi All,
I have a question regarding adapter trimming process of small RNA-seq data. The library for this dataset was prepared using NEBNext multiplex small RNA sample prep set for illumina (E7300S/L: https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale7300.pdf). So I used `bbduk.sh` from BBtools(https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/bbduk-guide/) using the following command:

bbduk.sh -Xmx1g in=Ago2_SsHV2L_1_CATGGC_L003_R1_001.fastq out=/media/owner/7ef86942-96a5-48a7-a325-6c5e1aec7408/trimmed_files/bbmap_trimmed/clean_Ago2_SsHV2L_1_CATGGC_L003_R1_001.fastq ref=NEB-SE_5_and_3_Prime.fa ktrim=r k=23 mink=11 hdist=1 tpe tbo

The adapter file`NEB-SE_5_and_3_Prime.fa` contains both 5' and 3' adapters:

>NEB_sRNA_read_1
AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC
>NEB_sRNA_read_2
AGATCGGAA

So the problem I have is with the trimmed file- the trimmed file now got rid of first adapter:

cat clean_Ago2_SsHV2L_1_CATGGC_L003_R1_001.fastq | head -n 20000 | grep AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC
owner@owner-HP-Z840-Workstation[bbmap_trimmed]

but it is still showing the second adapter:

owner@owner-HP-Z840-Workstation[bbmap_trimmed] cat clean_Ago2_SsHV2L_1_CATGGC_L003_R1_001.fastq | head -n 1000 | grep AGATCGGAA
TTTCTCTGAGCACTCCTTAGTACAAGATCGGAAGAGCACACGTCGAACTC
AAATGTTCTGAGGACTGGTTCTAGATCGGAAGAGCACCGTCTGAACTCCA
GATGGGCCCCGGGTTCGATTCCCGGCGAACGCACCAGATCGGAAGAGCCA
TTGGACGTGTTATTTTCAGACAAGATCGGAAGAAGCACACGTCTGAACTC

Can someone please help me understand if I need to remove both of these adapters in order to perform downstream/expression analysis? I have been using btrim to trim adapters from RNAseq data (in this case I never had to provide adapter infile), but this is the first time I am doing it with bbmap (and also with trimmomatic) for smallRNAseq data. In case of smallRNAseq data, do we normally trim both 5' and 3' adapters and have both adapter sequences in infile fortrimming? Can someone please help me understand this process? Thank you for your help in advance.

Answer: Hi:

I'm one of the developers of the NEBNext kits.
The reads you show seem to contain the sequence of the 3' adapter as expected for small RNA.
The 5' adapter sequence begins with a G (no A-tailing for this library type). The 5' adapter sequence should not be found in read 1, and I don't see it in the sequences you posted.

For our DNA Ultra*, RNA Ultra* and Small RNA methods, read 1 should always be trimmed with

AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC

(note: this is not always true for other vendor's methods)

For the Small RNA kit, Read 2 (if present, not typically done with short inserts) should be trimmed with

GATCGTCGGACTGTAGAACTCTGAACGTGTAGATCTCGGTGGTCGCCGTATCATT

The sequences of oligos used in our kits are documented at the end of our manuals.

I recommend using a simple program like flexbar [1] for single end trimming.

For paired end reads, presence of true adapter sequence requires that the insert is shorter than read length.
In that scenario, both read 1 and read 2 contain information about the position of the adapter.
Use of an adapter trimmer that takes advantage of this additional signal is advisable.
E.g. seqprep[2], flexbar (-ap option)[1], etc.

[1] Roehr, J. T., Dieterich, C., & Reinert, K. (2017). Flexbar 3.0 - SIMD and multicore parallelization. Bioinformatics, 33(18), 2941–2942. http://doi.org/10.1093/bioinformatics/btx330
https://github.com/seqan/flexbar

[2] https://github.com/jstjohn/SeqPrep