Nextseq 500 550 high output kit v2.5 300 cycles

TP53 gene mutation analysis was performed using a KAPA HyperChoice customized Next Generation Sequencing (NGS) panel (Roche Diagnostic Spa, Monza, Italy).
Samples libraries were prepared from 300 ng of DNA input using the KAPA HyperCap FFPET DNA workflow v1.1 according to the manufacturer’s instructions (Roche Diagnostic Spa, Monza, Italy). The quantity and quality of the libraries were checked with a QuBit dsDNA HS Assay kit (Thermo Fisher Scientific, Milan, Italy) and a High Sensitivity D1000 ScreenTape Assay kit (Agilent Technologies, Milan, Italy), respectively. Enriched libraries were pooled and sequenced with NextSeq^TM 550 using a NextSeq 500/550 High Output Kit v2.5 (300 cycles) (Illumina, Milan, Italy).
Reads obtained were aligned to the reference genome and sequencing data were validated with JuliaOmix^TM software v2.21.0 (GenomeUp, Rome, Italy).

DNA of the captured libraries was quantifed and quality verified for sequencing by QuantiFluor® dsDNA System (Promega Madison (Wis), USA) and Bioanalyzer High Sensitivity DNA Analysis System (Agilent Technologies, Inc. Santa Clara (CA), USA), respectively. Shotgun sequencing of the captured libraries was performed using a Nextseq 500 Illumina platform (Illumina Inc. San Diego (CA), USA) and ran with a NextSeq 500/550 High Output Kit v2.5 (300 Cycles), paired-end: R1:150; R2:150.

Total nucleic acids were extracted using a phenol/chloroform/isoamylalcohol protocol [40 (link)]. The extracts were subsequently treated with DNase to remove DNA (DNase I, Zymo Research, Freiburg, Germany). RNA concentrations were measured with the Qubit RNA HS Assay Kit (Qubit3.0 Fluorometer, Invitrogen, Waltham, MA, USA.). RNA extracts were cleaned with the MEGAclear kit (Thermo Fisher Scientific, Waltham, MA, USA); the quality of the RNA was verified by agarose gel electrophoresis and bioanalyzer (2100 Bioanalyzer, Agilent, Santa Clara CA, USA). We enriched the mRNA fraction and diluted inhibitory substances in the RNA extracts using the MessageAmp II-Bacteria RNA Amplification Kit (Thermo Fisher Scientific, MA, USA, input: 12.5 ng RNA). This method was previously validated for the preparation of metatranscriptomes [55 (link)]. Sequencing libraries were prepared with NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA; input 60 ng). Manufacturer’s instructions were followed except for Step 4, where fragmentation time was adjusted to 3 min and a size selection step with HighPrep PCR beads (MagBio Genomics Inc., Gaithersburg, USA) was introduced (desired insert size 250 bp). Libraries were paired-end sequenced with a NextSeq 550 System using the NextSeq 500/550 High Output Kit v2.5 (300 Cycles) (Illumina, San Diego, CA, USA).

The Pooled Amplicon Libraries from 8 DNA and 8 RNA libraries were loaded on a NextSeq 500/550 High Output Kit v2.5 (300 Cycles) and paired-end (2 × 101 bp) sequenced on a NextSeq500 instrument (Illumina). Data were analyzed with the TSO500 Local App v2.0 (Illumina) according to the user guide (TruSight Oncology 500 v2.0 Local App User Guide (1000000095997) (illumina.com; accessed on 5 May 2022)). It is important to note that deduplication is performed based on UMIs to remove all duplicate reads. Therefore, the indicated coverages are based on the unique reads only. DNA and RNA degradation was checked by calculating the mean amplicon insert size and deamination by analysis of the frequency of C > T; G > A changes. TMB was calculated as the total number of somatic non-hotspot variants with VAF > 5% per Mb of interrogated sequence. For MSI analysis, at least 40 MSI sites need to be interrogated. All passed-filter data including TMB, MSI, gene amplifications, splice variants, fusions, and small variants (SNVs and indels) are provided in the CombinedVariantOutput.tsv file of each sample. Only these data were considered for validation.