Dsdna br assay

Total genomic DNA was extracted from the whole body of 5th instar nymph using CTB Tissue Extraction Kit according to the manufacturer’s protocol (Intron, Seongnam-Si, Gyeonggi-do, Korea) after being pulverized in liquid nitrogen. DNA quality was assessed by spectrophotometric absorbance in NanoDrop 2000 Spectrophotometer V1.0 (Thermo Fisher Scientific, Wilmington, DE, USA) and Qubit dsDNA BR Assay, followed by electrophoresis on 0.8% agarose gel. In total, ≈2 μg of gDNA extracted from a pool of 15 bed bugs nymphs was used for 1/16th of a 70-mm × 75-mm Titanium PicoTiter plate of sequencing on a Roche 454 GS FLX sequencer with titanium chemistry (Roche Applied Science, Penzberg, Germany), performed by Macrogen Inc., South Korea. Sample preparation and analytical processing of sequence reads were performed according to manufacturer’s protocol for the Titanium series.

Five hundred nanogram of genomic DNA was fragmented (average size distribution ~500 bp, LE220, Covaris Inc), purified, libraries prepared (Agilent SureSelect XT custom kits, Agilent Technologies), and index tags applied (Sanger 168 tag set). Index tagged samples were amplified (6 cycles of PCR, KAPA HiFi kit, KAPA Biosystems), quantified (dsDNA BR assay, HS assay, Thermo Fisher Scientific), normalized (~0.85 ng/μl), then pooled together in an equivolume fashion. Pooled samples were submitted to cluster formation for HiSeq ×10 sequencing (32 lanes, 150 bp PE read length, Illumina Inc). The average sequencing coverage is 15-fold for all samples given that HAP1 is a haploid cell line. The details of sequence coverage for all clones and subclones are provided in Supplementary Data 5.

Isolation of genomic DNA from blood of all included patients was performed using FlexiGene DNA kit (Qiagen, Hilden, Germany) or Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
FFPE blocks of normal colorectal mucosa and CRC tissue were obtained when available. For each FFPE specimen, 10-20 x 10-μm sections were cut from a single representative block per case, using macrodissection with a scalpel if needed to enrich for tumor cells. After deparaffinization using the Qiagen Deparaffinization Solution (Qiagen), DNA was isolated using the QIAmp DNA FFPE Tissue Kit (Qiagen) according to manufacturer’s instructions.
DNA quality was tested using a NanoDrop ND 1000 Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA), electrophoresis in agarose gel and a Qubit Fluorometer (Invitrogen, Carlsbad, CA, USA) using the dsDNA BR Assay (Invitrogen, Carlsbad, CA, USA).

Genomic DNA was extracted from adipose fin clips using a column extraction kit (NucleoSpin Tissue, Macherey Nagel, Düren, Germany) following the manufacturer’s recommended protocols. DNA quality was assessed by visualization after gel electrophoresis on a 2% Agarose gel and quantified using a Qubit 2.0 Fluorometer with the dsDNA BR Assay (Life Technologies, Carlsbad, CA, USA). Each sample was normalized to a total amount of 1 µg of DNA with a minimum concentration of 25 ng/µL. DNA was used to construct a single ddRAD-library containing 44 individuals, following a previously described protocol in [67 (link)] with a modified set of barcoded P1 and P2 adapters for paired-end sequencing on Illumina platforms. In brief, each sample was digested using two restriction enzymes, a rare-cutting enzyme (PstI-HF, 20 units) and a frequent-cutting enzyme (MspI, 20 units) (New England Biolabs, Ipswich, MA, USA). Samples were individually barcoded using unique combinations of barcoded P1 and P2 adapters. After multiplexing of the barcoded samples, a size selection step was performed using the Pippin Prep (Sage Science, Beverly, MA, USA) and fragments between 145–295 bp were selected. The amplified library was sequenced on half a lane (200 million reads) of an Illumina NextSeq 500 (75 bp paired-end reads) at Glasgow Polyomics (University of Glasgow).

DNA was extracted from fin clips and muscle tissue using the NucleoSpin Tissue kit (Macherey-Nagel), following the manufacturers recommendations. DNA quality and quantity were assessed using agarose gel electrophoresis and the Qubit Fluorometer with the dsDNA BR Assay (Life Technologies). ddRADseq libraries were prepared using a modified version of the Recknagel et al. (2015) [79 (link)] ddRADseq protocol for Illumina sequencing platforms. Paired-end 75-bp sequencing was performed on the Illumina NextSeq500 platform at Glasgow Polyomics (University of Glasgow) at 3-4M read coverage per individual.