miRNA identification, mapping and quantification
Detection of known and novel miRNAs was done using miRDeep2 (Friedländer
et al., 2008; Friedländer, Mackowiak, Li, Chen, & Rajewsky, 2012).
First, reads from all samples were merged and mapped to a P. napigenome (Lohse, Hayward, Ebdon, of Life, & Consortium, 2021). Reads were
compared to known miRNAs from all mature miRNAs and hairpin sequences
from B. mori and all mature reads from Heliconius
melpomene from miRbase v22 (Kozomara, Birgaoanu, & Griffiths-Jones,
2019). Remaining unmapped sequences were then used to identify novel
miRNAs and their hypothetical secondary structure (Friedländer et al.,
2012), giving an output of known and novel miRNAs in all samples in the
data set. Identified miRNAs were then discarded if they did not meet
threshold requirements of a miRDeep2 score ≥ 1, presence in more than 5
samples, and a total read coverage of ≥ 10 reads across samples. All
identified miRNAs were then used to obtain read counts for each miRNA
for each sample using the quantifier.pl script in miRDeep2.