Kapa library quantification kit abi prism

Total RNA was extracted from the plasma samples using TRIzol (ThermoFisher, Waltham, MA, USA). Each sample was purified using miRNAeasy mini kit (Qiagen, Germantown, MD, USA) according to manufacturer’s instructions. RNA quality was measured using a Nanodrop spectrophotometer (ND-1000, Nanodrop Technologies, Waltham, MA, USA). RNA integrity was determined by gel electrophoresis. miRNA concentrations were measured by a Qubit RNA HS Assay on a Qubit fluorometer (ThermoFisher, Waltham, MA, USA). A small RNA library preparation was performed following the NEB NEBNext Multiplex Small RNA Library Prep Set for Illumina protocol small by The Centre for Applied Genomics at The Hospital for Sick Children (Toronto, ON, Canada). RNA libraries were quantified by qPCR using the Kapa Library Quantification Illumina/ABI Prism Kit protocol (KAPA Biosystems, Boston, MA, USA). Libraries were pooled in equimolar quantities and single-end sequenced on Rapid Run Mode flow cell with the V3 sequencing chemistry on an Illumina HiSeq 2500 platform following Illumina’s recommended protocol to generate single-end reads of 50-bases in length.

By adding sample specific index sequences to the amplicons, pools of several samples are made for sequencing. Each individual library, with six target amplicons, is quantified using KAPA Library Quantification Kit—Illumina/ABI Prism (KAPA biosystems) qPCR, and pooled in equal molar concentrations. Twenty-five individual libraries were pooled together for sequencing to target at least 100,000x coverage, allowing for extremely low-level detection of resistant alleles. At least 25% of each sequencing run was filled with whole genome or PhiX control samples to ensure base diversity and reduce complications with sequencing. For the validation a single sequencing pool was sequenced on the Illumina MiSeq platform using 2x300bp version 3 sequencing chemistry (Illumina). If PhiX control was not used for base diversity (e.g., at 25% of a sequencing run), then it was spiked in a low concentration (i.e., 1–5%) in each run for overall error rate examination. In all runs the pure susceptible isolate was run as a SMOR error control for the analysis. Novel Read 1, Read 2, and indexing sequencing primers were used for sequencing (S3 Table) and were added for a final concentration of 0.5 uM as per Illumina’s recommendation. All sequencing read files were deposited in NIH Short Read Archive (Bioproject # PRJNA271805).