Agencourt ampure xp kit

DNA isolation and targeted sequencing were performed in Burning Rock Biotech, a commercial clinical laboratory accredited by the College of American Pathologist (CAP) and certified by the Clinical Laboratory Improvement Amendments (CLIA), according to optimized protocols as described previously [15 (link), 16 (link)]. Briefly, tissue DNA was extracted from formalin-fixed, paraffin-embedded tumor tissues using QIAamp DNA formalin-fixed paraffin-embedded tissue kit (Qiagen, Hilden, Germany) and circulating cell-free DNA (cfDNA) was extracted from 4–5 ml of plasma samples using a QIAamp Circulating Nucleic Acid kit, according to the manufacturer’s standard protocol (Qiagen, Hilden, Germany). Fragments between 200 and 400 bp from the sheared tissue DNA and cfDNA were purified (Agencourt AMPure XP Kit, Beckman Coulter, CA, USA), hybridized with capture probes baits, selected with magnetic beads, and amplified. Target capture was performed using a commercial panel consisting of 520 genes (OncoScreen Plus), spanning 1.64 megabases of the human genome. The quality and the size of the fragments were assessed by high sensitivity DNA kit using Bioanalyzer 2100 (Agilent Technologies, CA, USA). Indexed samples were sequenced on Nextseq 500 (Illumina, Inc., CA, USA) with paired-end reads and an average sequencing depth of 1,000 × for tissue samples and 10,000 × for liquid biopsy samples.

We designed LR-PCR primers to amplify DNA fragments by PCR. The LR-PCR products were purified using the Agencourt AMPure XP kit (Beckman Coulter, Inc., Brea, CA, United States), followed by quantification and fragmentation using a NEBNext Fast DNA Fragmentation Kit (New England BioLabs, Ipswich, MA). Sequencing was performed with an ABI3730xl sequencer (Applied Biosystems, United States).

Next-generation sequencing (NGS) was performed by Illumina MiSeq (Illumina). The tissue DNA was extracted from FFPE tumour tissues using QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany). Fragments between 200 and 400 bp from the sheared tissue DNA were purified (Agencourt AMPure XP Kit, Beckman Coulter, CA, United States), hybridized with capture probes baits, selected with magnetic beads, and amplified. Target capture was performed using a commercial panel consisting of 520 genes (Supplementary Table S2) chosen by Guangzhou Burning Rock Biotech Ltd. Sequence data were mapped to the reference human genome (hg19) using Burrows-Wheeler Aligner version 0.7.10. Local alignment optimization, duplication marking and variant calling were performed using Genome Analysis Tool Kit version 3.2, and VarScan version 2.4.3. Base calling in tissue samples required at least 8 supporting reads for single nucleotide variations (SNVs) and 2 and 5 supporting reads for insertion-deletion variations (Indels), respectively. Copy number variations (CNVs) were analyzed based on the depth of coverage data of capture intervals.

Library preparation and sequencing were performed by Vertis Biotechnology AG, Germany. Briefly, the total RNA samples were split into two, one was enriched for the small RNA fractions < 200 nt (s) specifically using the RNeasy MinElute Cleanup Kit (Qiagen). Ribosomal RNA molecules were depleted from both samples using the Ribo-Zero rRNA Removal Kit for bacteria (Epicenter). The rRNA depleted RNA fractions were first treated with Tobacco Acid Pyrophosphatase (TAP, Epicenter). Afterward, oligonucleotide adapters were ligated to the 5′ and 3′ ends of the RNA samples. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3′ adapter as primer. The resulting cDNAs were amplified by PCR using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. The cDNA pool was sequenced on an Illumina NextSeq 500 system using 75 bp read length.

Each sample was lysed in extraction buffer using a bead-mill homogenizer, and total DNA was extracted using a phenol-chloroform protocol. In the case of roots and leaves, we did not surface sterilize samples, so we characterized both endophyte and epiphyte communities. After quality check, we prepared libraries targeting the bacterial 16 S rRNA gene (region V3-V4) using the primer pair 515 f/806rB^{32 (link)} (~350 bp amplicon size). Amplifications were also carried out on DNA extracted from the soil inoculum and non-template controls, where the sample was replaced with nuclease-free water to account for possible contamination of instruments, reagents, and consumables used for DNA extraction. After this first PCR, samples were purified (Agencourt AMPure XP kit, Beckman Coulter) and used for a second short-run PCR to ligate Illumina adaptors. Libraries were then purified again, quantified using a Qubit spectrophotometer (Thermo Fisher Scientific Inc.), normalized using nuclease-free water, pooled together, and sequenced on an Illumina NovaSeq 6000 SP 250PE flow cell at the Genomic Sciences Laboratory of North Carolina State University (Raleigh, NC, USA).