Kapa hyper prep kit

Indexed Illumina next-generation sequencing (NGS) libraries were prepared from plasma DNA. Plasma DNA was used for library construction without additional fragmentation. Genomic DNA was sheared before library construction using a Covaris S2 instrument (Woburn, MA, USA) to obtain 200-bp fragments. According to the manufacturer's protocol, NGS libraries of plasma DNA were constructed using the KAPA Hyper Prep Kit (Kapa Biosystems, Wilmington, MA, USA). A sequencing library was prepared using the KAPA Hyper Prep Kit (Kapa Biosystems) and SureSelect Target Enrichment System (Agilent Technologies). End repair and A-tailing reactions were performed in 60-µL reaction volumes. The mixtures were then incubated at 20 °C and 65 °C for 30 min each. Adapter ligation was performed using 110-µL volumes, and samples were incubated at 16 °C for 16 h using a SureSelect Adapter (Agilent Technologies). After postligation cleanup, the ligated fragments were amplified in a 50-µL solution containing 2 × KAPA HiFi HotStart ReadyMix and 10 × KAPA Library Amplification Primer Mix (Kapa Biosystems). We used the following cycling protocol: 98 °C for 45 s, 14–16 cycles (depending on the input DNA mass) of 98 °C for 15 s, 65 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min (1 cycle). Library purity, library concentration, and fragment length were determined using a 2100 Bioanalyzer (Agilent Technologies).

Indexed Illumina NGS libraries were prepared from plasma, tumor, and germline DNA. Plasma DNA was used for library construction without additional fragmentation. Tumor and germline genomic DNA were sheared before library construction with a Covaris S2 instrument (Woburn, MA, USA) to obtain 200-bp fragments. The NGS libraries of plasma DNA were constructed using the KAPA Hyper Prep Kit (Kapa Biosystems, Wilmington, MA, USA), following the manufacturer's protocols. A sequence library was prepared using a combination of the KAPA Hyper Prep Kit (Kapa Biosystems) and the SureSelect Target Enrichment System (Agilent Technologies, Santa Clara, CA, USA). End repair and A-tailing reactions were performed in 60-μL reaction volumes. The mixtures were incubated at 20°C for 30 minutes and 65°C for 30 minutes. Adapter ligation was performed using 110 μL and samples were incubated at 16°C for 16 hours using Agilent SureSelect Adapter. After post-ligation cleanup, the ligated fragments were amplified in 50 μL containing 2× KAPA HiFi HotStart ReadyMix and 10× KAPA Library Amplification Primer Mix. The following cycling protocol was used: 98°C for 45 s; 14–16 cycles (depending on the input DNA mass) of 98°C for 15 s, 65°C for 30 s, and 72°C for 30 s; and 1 cycle of 72°C for 5 min. Library purity, library concentration, and fragment length were determined using a 2100 Bioanalyzer (Agilent).

Genomic DNA extraction and purification were performed with the DNeasy Blood & Tissue Kit (Qiagen) from white blood cells or the QIAamp DNA FFPE Tissue Kit (Qiagen) from formalin-fixed paraffin-embedded (FFPE) samples, which was then quantified by a Qubit Fluorometer (Life Technologies) with the dsDNA HS Assay Kit. Sequencing libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems), As described previously [17 (link)], the indexed DNA libraries for sequencing were prepared (KAPA Hyper Prep Kit, KAPA Biosystems) and captured by probe-based hybridization, which targeted over 400 cancer-related genes and over 12,000 SNPs that evenly distributed throughout the whole genome. The Illumina HiSeq4000 platform was used for DNA sequencing.

Two-week-old Arabidopsis seedlings of Col-0 wild type and trb1/2/3 triple mutans were used for DNA extraction using DNeasy Plant Mini Kit (QIAGEN). A total of 500 ng DNA was sheared with Covaris S2 (Covaris) into around 200 bp at 4 °C. The DNA fragments were used to perform end repair reaction using the Kapa Hyper Prep kit (Roche), and together with TruSeq DNA Sgl Index Set A/B (Illumina) to perform adapter ligation. The ligation products were purified with AMPure beads (Beckman Coulter), and then converted with EpiTect Bisulfite kit (QIAGEN). The converted ligation products were used as templates, together with the primers from the Kapa Hyper Prep kit (Roche) and MyTaq Master mix (Bioline), to perform PCR. The PCR products were purified with AMPure beads (Beckman Coulter) and sequenced by an Illumina NovaSeq 6000 sequencer.

For each sample, 120 ng of genomic DNA in 60 μL of 10 mM Tris-HCl, pH 8.0 buffer, was placed into 1.5 mL RNase-/DNase-free, low binding microcentrifuge tubes. Library creation steps were performed by the IIHG Genomics Division, and included DNA shearing using the Covaris Adaptive Focused Acoustics^TM process (Covaris E220 Focused-ultrasonicator; Covaris, Inc., Woburn, MA, United States), and DNA fragment purification and end polishing (KAPA Hyper prep kits; Kapa Biosystems, Inc., Wilmington, MA, United States) prior to ligation to indexed adaptors. The library size distribution was validated using the Agilent 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA, United States), and quantified using the q-PCR KAPA library amplification module following manufacturer instructions (Kapa Biosystems, Inc.). The indexed libraries were normalized, pooled, and clustered on a flow cell using the cBOT Cluster Generation System (Illumina, Inc., San Diego, CA, United States) and sequenced on the Illumina HiSeq 4000 System (Illumina, Inc.) in high output mode (1 lane, 2 × 150 bp). FASTQ files are accessible at ENA (Study Accession: PRJEB23134) and NCBI repositories (BioProject ID: PRJNA414922), and MG-RAST contains QA/QC and analyses of metagenomes (MG-RAST project mgp21252).

To extract DNA, we transferred nematodes from three recently starved 10 cm NGMA plates originally spotted with OP50 E. coli into a 15 ml conical tube by washing with 10 ml of M9. We then used gravity to settle animals in the conical tube, removed the supernatant, and added 10 ml of fresh M9. We repeated this wash method three times to serially dilute the E. coli in the M9 and allow the animals time to purge ingested E. coli. Genomic DNA was isolated from 100 to 300 µl nematode pellets using the Blood and Tissue DNA isolation kit (cat no. 69506, Qiagen) following established protocols (Cook et al., 2016 (link)). The DNA concentration was determined for each sample using the Qubit dsDNA Broad Range Assay Kit (cat no. Q32850, Invitrogen). Sequencing libraries were either generated with KAPA Hyper Prep kits (Kapa Biosystems), Illumina Nextera Sample Prep Kit (Illumina, Inc.), or New England BioLabs NEBNext Ultra II FS DNA Library Prep (NEB). Samples were sequenced at the Duke Center for Genomic and Computational Biology, Novogene, or the Northwestern Sequencing facility, NUSeq. All samples were sequenced on the Illumina HiSeq 4000 or NovaSeq 6000 platform (paired‐end 150 bp reads). The raw sequencing reads for strains used in this project are available from the NCBI Sequence Read Archive (Project PRJNA549503).

Single‐cell transcriptome sequencing libraries were prepared according to the previously published methods.¹⁵ Human gastric glands and cultured cells were dissociated into single cell suspensions and sorted into 96‐well plates containing 2 μl lysis buffer. SuperScript II reverse transcriptase (18064014, Invitrogen), template switch oligo primer, and barcoded primers with unique molecular identifiers (UMIs) were used to reverse transcribe mRNAs, followed by cDNAs amplification and purification. After being amplified using a biotin‐anchored primer with index tags, cDNAs were fragmented and enriched for library construction. The final libraries were constructed by KAPA Hyper Prep Kits (KK8504, KAPA Biosystems), and sequenced on Illumina HiSeq 4000 platform or Illumina NovaSeq 6000 platform (Novogene or Anoroad).