Qubit 3.0 system

High molecular weight (HMW) DNA was extracted from the specimens following an adaptation of the salting-out method of Miller et al. (1988) (link) (10x Genomics 2018 ). Before extraction, the genitalia of the individual males was removed and stored as species vouchers in 99.5% ethanol at –20°C. The remainder of each insect was homogenized using sterile scalpel blades, and then incubated overnight at 37°C in 600 µl lysis buffer (10 mM Tris–HCl, 400 mM NaCl, and 100 mM EDTA, pH 8.0) with 100 µl of Proteinase K (20 mg/ml). Genomic DNA was then salted out by adding 240 µl of 5 M NaCl and cleaned using 70% ethanol. Finally, the extracted HMW DNA was quantified using the Qubit 3.0 system (Invitrogen) and the size distribution (>20 kbp) was confirmed by visualization on a 0.8% Agarose gel alongside a 1Kb extension ladder (Invitrogen).

The fragment DNA was generated with Bioruptor^® (Diagenode,Bioruptor UCD-200) following the manufacturer’s instructions. Libraries were constructed using the KAPA HyperPrep DNA Library Kit (KAPA Biosystem, KK8504). Dual-indexed sequencing libraries were amplified by polymerase chain reaction (PCR) with KAPA HiFi HotStart ReadyMix (KAPA,KK2602) for 4–6 cycles, then cleaned up by purification beads (Corning, AxyPrep FragmentSelect-I Kit, 14223162). Library concentration and quality were determined by the Qubit™ 3.0 system (Invitrogen) and the Bioanalyzer 2100 (Agilent, Agilent HS DNA Reagent, 5067–4627).

The fecal samples (50 mg) were weighed in 1 ml microcentrifuge tubes and placed in liquid nitrogen and were subsequently stored at -80℃ until used. Total DNA was extracted from frozen fecal samples for metagenomic sequencing using a QIAamp Fast DNA stool Mini Kit (QIAGEN, Inc., Germany). The whole extraction process was performed as instructed by the manufacturer. Agar-gel electrophoresis (AGE), Nanodrop, and Qubit 3.0 system (Thermo Fisher Scientific, Inc., America) were used to determine the purity and integrity of the extracted DNA. DNA of sufficient purity and integrity were tested for library construction and sequenced using an Illumina HiSeq high-throughput sequencing platform (Qinglian Biotec. Co., Ltd, Beijing, China) along with a KAPA Hyper Prep Kit (KAPA Biosystems, Inc., America). The raw data obtained from the sequencing were further filtered to obtain a higher quality of clean reads for subsequent informational analysis. SOAPdenovo Assembly software was used to assemble the Clean Data and Scaftigs were obtained^{69 (link)}. The scaftigs were further filtered for statistical analysis and subsequent genetic prediction (fragments cut-off: 500 bp).

DNA for WES was isolated from FFPE tissues after microdissection, deparaffinization and proteinase K digestions using the Maxwell RSC16 extraction system according to the instructions of the manufacturer (Promega, Madison, WI, USA). DNA concentration was measured fluorimetrically by the Qubit 3.0 system (Thermo Fisher Scientific, Waltham, MA, USA) and the DNA quality was determined by a qPCR assay (RNAse P assay, Thermo Fisher Scientific) as described (Endris et al. 2013 (link)).
The DNA degradation index (DDI) was calculated by dividing the quantity of amplifiable DNA calculated by the RNAse P assay by the concentration of the total amount of DNA quantified by the Qubit dsDNA high-sensitivity assay. Tumors with DDI values < 0.2 were not used for sequencing.

After marking of the tumor area and annotation of percentage of vital tumor tissue (≥ 50% tumor cell content) for micro-dissection, DNA was extracted using the Maxwell 16 RSC extraction system (Promega) according to the manufacturer´s protocols. DNA concentration was measured fluorometrically using the QuBit 3.0 system (Thermo Fisher Scientific) and DNA quality was determined by a qPCR assay (RNAseP assay, Thermo Fisher Scientific).