Hiseq rapid sbs kit v2 200 cycles

RNA Samples were assessed for quality with an Advanced Analytics Fragment Analyzer (High Sensitivity RNA Analysis Kit – Catalog # DNF-491 / User Guide DNF-491-2014AUG13) and quantity with a Qubit RNA quantification kit (Qubit® RNA HS Assay KitAssay Kit – Catalog # Q32852). Given satisfactory quality and quantity, samples were used for library builds with the standard Kapa Biosystems mRNA HyperPrep Kit (KapaBiosystems KAPA mRNA HyperPrep Kit – Catalog # KK8540 / KapaBiosystems mRNA HyperPrep Kit TDS KR1352 – v4.17). Upon library build completion, samples were assessed for quality and average fragment size with the Advanced Analytics Fragment Analyzer (High Sensitivity NGS Analysis Kit – Catalog # DNF-486 / User Guide DNF-486-2014MAR10). Quantity was assessed with an Illumina Universal Adaptor-specific qPCR kit from Kapa Biosystems (Kapa Library Quantification kit for Illumina NGS – Catalog # KK4824 / KAPA Library Quantification Technical Guide - AUG2014).
After final library QC was completed, samples were equimolar-pooled and clustered for sequencing on the HiSeq2500 machine. The sequencing run was performed using Illumina HiSeq Rapid SBS v2 chemistry (HiSeq Rapid SBS Kit v2 200 cycles - Catalog # FC-402-4021, HiSeq PE Rapid Cluster Kit v2 - Catalog # PE-402-4002), and data were sent to UAGC Biocomputing Group for further analysis and transmission to the client.

Starting with 500 ng of total RNA, cDNA library templates were synthesized using the Illumina TruSeq® Stranded Total RNA LT (Set A) Kit with Ribo-Zero Globin rRNA reduction, as described in the TruSeq® Stranded Total RNA Sample Preparation Guide (Rev. E., October 2013). Libraries were validated using the HS D1000 ScreenTape on the Agilent 2200 TapeStation and quantified on the Qubit® 2.0 Fluorometer by the Qubit® dsDNA HS Assay (ThermoFisher). The Illumina HiSeq2500 instrument was used to cluster the samples onto the flow cell. The pools were put on at 10 pM and run in rapid mode at 2 × 100 paired end. The Illumina HiSeq Rapid PE Cluster Kit V2 and HiSeq Rapid SBS Kit v2 200 cycles were used.

The Whole Genome Sequence (WGS) analysis for BAC 86/19 was conducted at the NGS multiuser platform of Oswaldo Cruz Foundation (FIOCRUZ), located in Rio de Janeiro, Brazil. First, DNA quantification was performed using the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and the Agilent High Sensitivity DNA Kit (Agilent, Santa Clara, CA, USA). The WGS procedure was carried out on a HiSeq instrument (Illumina, San Diego, CA, USA) using HiSeq Rapid SBS Kit v2 (200 cycles) chemistry and the Nextera DNA Flex Library preparation kit (Illumina, San Diego, CA, USA) following the manufacturer’s guidelines.
The resulting reads were deposited in SRA (Sequence Read Archive), NCBI (National Center for Biotechnology Information) under accession number SRR19621772.