RAD-seq library preparation, sequencing and read filtering
Restriction-site-associated DNA libraries of 519 ABFT individuals were
prepared following Etter et al. (2012)
. Input DNA (ranging from 50 to
500 ng) was digested with the SbfI restriction enzyme and ligated
to modified Illumina P1 adapters containing 5bp unique barcodes. Pooled
DNA of 32 individuals was sheared using the Covaris® M220
Focused-ultrasonicator™ Instrument (Life Technologies) and size selected
to 300-500 pb on agarose gel. After Illumina P2 adaptor ligation, each
library was amplified using 14 PCR cycles. Each pool was paired-end
sequenced (100 pb) on an Illumina HiSeq2000.
Demultiplexing, quality filtering
(removing reads whose average Phred score is lower than 20 and
truncating them to 90 nucleotides to remove low-quality bases at the
end) and PCR duplicate removal were preformed using theprocess_radtags and clone_filter modules ofStacks version 2.3e (Rochette et
al. 2019).