A total of 41,603 SNP were identified between the cold sensitive and tolerant genotypes with minimum read of four.
Low quality nucleotide reads trimming (Phred quality threshold of 25 and minimum reads length of 40 nt) was performed with Sickle, a sliding-window, adaptive, quality-based trimming tool for FastQ files (available at [ 95]).
The default assembly options of minimum read length of 40 nucleotides, minimum sequence overlap of 40 nucleotides and a minimum relative overlap score of 80% of similarity were used.
All KvarQ results were generated using default parameters, i.e. a quality cutoff of 13, a minimum read length of 25, a minimum overlap of 25, a minimum coverage of two reads for allele calls and a maximum of two errors per read.
Adaptor sequences were trimmed, and low-quality reads were filtered out using the following parameters: ambiguous limit of 2; quality limit of 0.05; minimum read length of 50 nt.
a contigs were selected with minimum length of 300-bp and minimum mapped reads of 50.
Vcfutils.pl varFilter was applied under default settings to remove low quality SNPs, with the addition of a minimum read depth of 10 [ 105].
Further, reads were trimmed using 'Trim the reads by quality' (version 1.2.2) tool (Phred quality cutoff of 20 and minimum read length of 40 nucleotides).
Using the matched L2-poly(A) samples, a pseudogene was called expressed if it passed a minimum array intensity threshold of 100 or minimum read count of 1.
For each library, sequences were quality trimmed allowing for 1 ambiguous nucleotide, a quality score limit of 0.05, and minimum read length of 15 nucleotides.
