Qubit dsdna hs kit

40 ng of each plasmid DNA was sheared into approximately 350 base pair in length by sonication using a Covaris Sonicator LE220 (Covaris). Fragmented DNA was uniquely index tagged with NEBNext Multiplex Oligos for Illumina (New England Bio-Labs, E7780S and ES7600S). The Kapa LTP Library Preparation Kit (KAPA Biosystems, Roche7961880001) was deployed in this process. Libraries were quantified and quality controlled with Qubit dsDNA HS kit (ThermoFisher) and Agilent 4200 Tapestation System (Agilent). Equimolar amounts of each library were pooled together and sequenced on the Illumina MiSeq platform using MiSeq Reagent Micro Kit v2 (2x 150-cycles). Plasmid sequences were assembled using SPAdes v3.10.1 with multiple k-mer sizes. Minimum depth of 100 reads and Phred quality of 30 were used for consensus calling of the assembled sequences.

RNA-seq libraries were constructed using NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770) following the manufacturer’s instructions. Briefly, mRNA isolated from ∼1 μg of total RNA was fragmented to an average size of 200 nt by incubating at 94° for 15 min in first strand buffer, cDNA was synthesized using random primers and ProtoScript II Reverse Transcriptase followed by second strand synthesis using NEB Second Strand Synthesis Enzyme Mix. Resulting DNA fragments were end-repaired, dA tailed and ligated to NEBNext hairpin adaptors (NEB #E7335). After ligation, adaptors were converted to the ‘Y’ shape by treating with USER enzyme and DNA fragments were size selected using Agencourt AMPure XP beads (Beckman Coulter #A63880) to generate fragment sizes between 250 and 350 bp. Adaptor-ligated DNA was PCR amplified followed by AMPure XP bead clean up. Libraries were quantified with Qubit dsDNA HS Kit (ThermoFisher Scientific #Q32854) and the size distribution was confirmed with High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies #5067- 4626). Libraries were sequenced on Illumina HiSeq2500 in single read mode, with an approximately similar depth of 30 million reads per sample, and a read length of 50 nt following manufacturer’s instructions. Base calls were performed with RTA 1.18.64 followed by conversion to FASTQ with bcl2fastq 1.8.4.

DNA from environmental samples was extracted using the DNeasy PowerSoil Pro Kit (QIAGEN, Germany) according to the manufacturer’s protocol. The concentration of the extracts was measured using the Qubit dsDNA HS kit (Thermo Fisher Scientific, USA, #Q33231) with a Qubit 3.0 fluorometer (Thermo Fisher Scientific, USA), while DNA purity was assessed with a NanoDrop One Spectrophotometer (Thermo Fisher, USA). Sample DNA fragment sizes were inspected using an Agilent 2200 Tapestation system with Genomic DNA ScreenTapes (Agilent Technologies, USA, #5067-5365). DNA samples were also size-selected (with the exception of BRK sample due to limited DNA amounts) using the Circulomics SRE XS kit (Circulomics, USA), following the manufacturer’s instructions to deplete DNA fragments below 10 kb.

RNA-Seq libraries were prepared using the Universal Plus mRNA-Seq with NuQuant (NuGEN, cat no. MO1485). For each sample, 100 ng of RNA (in a volume of 50ul) was used for poly A enrichment. First- and second-strand cDNA was synthesized followed by adapter and unique index ligation. Samples were barcoded using the Universal Plus (UDI) 96-Plex Adaptor Plate (NuGEN, cat no. S02480). The concentration of each library was measured using a Qubit dsDNA HS kit (Thermo Fisher, cat no. Q32854). The correct size of each library was confirmed by Agilent Bioanalyzer analysis using the DNA High Sensitivity Kit (Agilent Technologies, cat no. 5067-4626). In addition, correct adapter and index ligation, as well as the library concentration, was validated by sequencing all libraries on a MiSeq Nano Kit V2 300 cycles (Illumina, cat. no. MS-103-1001). 1.5 pM of barcoded library was denatured, and sequencing was performed on an Illumina NextSeq 500 using the NextSeq 500 75 cycles High Output Kit v2.5 (Illumina, cat no. 20024906). One single-end replicate was performed for each sample. The MiSeq quality control run, as well as the data run on the Illumina NextSeq 500 yielded reads with a data quality of 94.9% of reads at or above Q30. The read alignment rate to the human reference genome Hg38 was 98.6% or higher for each sample.

To study phage virions, we isolated the faecal VLP fraction and sequenced dsDNA phages as previously described^{19 (link)}. Briefly, the VLPs were extracted from 500 mg of faeces using high-speed centrifugation followed by filtration through a 0.45 µm membrane. Any free-DNA debris was digested prior to lysing the VLPs, whereafter the DNA was purified using a two-step phenol/chloroform extraction protocol. Finally, the DNA was purified using the DNeasy Blood&Tissue kit (Qiagen Cat#69506) according to the manufacturer’s protocol. Library preparation was done with the NEBNext Ultra II FS DNA library prep kit (New England Biolabs Cat#E7805L) and the NEBNext Multiplex Oligos for Illumina dual indexes (New England Biolabs Cat#E7600S) according to manufacturer’s instructions. Quality and concentration of the VLP libraries were assessed with the Qubit dsDNA HS kit (ThermoFisher Cat#Q32854) and with the Agilent High Sensitivity D5000 ScreenTape system (Agilent Technologies). Libraries were sequenced using 2×150 bp paired-end chemistry on an Illumina NovaSeq 6000 platform with the S4 Reagent Kit v1.5, 300 cycles (Illumina Cat#20028312) at the Core Facility Genomics of the Amsterdam UMC.

Samples from 84 patients were analyzed. The cancerous areas were assessed and recovered using previously reported methods [19 (link)]. Chromosomal DNA was isolated from formalin-fixed paraffin-embedded (FFPE) colorectal adenocarcinoma samples using the QIAamp DNA FFPE Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. DNA concentration was determined by measuring the fluorescence using the Qubit dsDNA HS kit (Thermo Fisher Scientific, Waltham, MA, USA).