Abstract
A necessary pre-processing data analysis step is the removal of adapter sequences from the raw reads. While most adapter trimming tools require adapter sequence as an essential input, adapter information is often incomplete or missing. This can impact quantification of features, reproducibility of the study and might even lead to erroneous conclusions. Here, we provide examples to highlight the importance of specifying the adapter sequence by demonstrating the effect of using similar but different adapter sequences and identify additional potential sources of errors in the adapter trimming step. Finally, we propose solutions by which users can ensure their small RNA-seq data is fully annotated with adapter information.
Highlights
Small non-coding RNAs comprise short RNAs less than 200 nt in length, including microRNAs, Piwi-interacting RNAs and small nucleolar RNAs and which have a variety of functions
An Next generation sequencing (NGS) experiment requires the construction of a library, which includes ligation of an adapter that acts as a binding site for priming the sequencing reaction and capturing the endogenous small RNA inserts [1,2]
Due to the short insert size, the 3’ adapter sequence is commonly included in the raw data reads and removing the adapter from raw reads is a necessary data analysis step
Summary
Small non-coding RNAs comprise short RNAs less than 200 nt in length, including microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and small nucleolar RNAs (snoRNAs) and which have a variety of functions. The adapter sequence in the instruction manual for the NEBNext Small RNA Library Prep Kit for Illumina is specified as: 5’-rAppAGATCGGAAGAGCACACGTCT-NH2-3’
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