Target enrichment of Epermenia chaerophyllella ZFMK 41_S10
Genomic DNA has been extracted from museum material, repaired (NEBNext FFPE DNA Repair Mix), purified (AMPure XP beads) followed by end-repair and A-Tailing reactions (Agilent Sure Select XT2 Library Prep Kit). After adaptor-ligation (NEBNext Quick Ligation Module, NEBNext Multiplex Oligos for Illumina Kit) a pre-capture library amplification (NEBNext Multiplex Oligos for Illumina, NEBNext Q5 HotStart HiFi PCR Master Mix) was performed to dual-index the libraries. After PCR reaction samples were purified (AMPure XP beads). Enrichment and capture steps were performed (SureSelect XT2 protocol) with additional modifications. Specimens were pooled in equimolar absolute amounts in a pool of 8 samples. Hybridization was performed on each pool (SureSelect XT2 Pre-Capture ILM Module Box 2, SureSelect Custom Baits 6-11.9 Mb kit, protocol for captured library size >3.0 Mb with a volume reduced by 50%). Enriched libraries were captured (Dynabeads MyOne Streptavidin T1), washed (SureSelect XT2 Pre-Captured Box 1) followed by an elution of Dynabeads containing the selected DNA (nuclease-free water). Amplification of the captured libraries (Herculase II PCR mix and XT2 Primer) was followed by purification (AMPure XP beads). The resulting hybridized libraries were quantified with a Quantus Fluorometer and quality was checked with a Fragment Analyzer. Amplified libraries were then sequenced on an Illumina Nextseq 500 System.