Nextseq 550 high output kit

Whole-exome sequencing (WES) was performed on genomic DNA obtained from the patient’s peripheral blood as previously described [15 (link)]. The DNA library was prepared using the Illumina’s TruSeq DNA Exome Kit. Sequencing was performed on the NextSeq-500 platform using the NextSeq 550 High Output kit (150 cycles) according to the manufacturer protocol (Illumina, California, USA). Raw sequence reads were aligned to the human genome reference sequence hg19 using FastQC 0.11.7 and BWA (Aligner) 0.7.15. Imported variants were annotated, filtered, and classified using VariantStudio (Illumina, California, USA). The variants were further filtered using the dbSNP (https://www.ncbi.nlm.nih.gov/snp/) and the Genome Aggregation Database (https://gnomad.broadinstitute.org/). Sanger sequencing was performed to confirm the presence of the NGS-derived variant in the patient. Familial segregation analysis by Sanger sequencing was limited to the patient’s non-affected brother due to the unavailability of parents or other family members.

The final amplified libraries were purified using Agencourt XP beads and quantified using the Qubit dsDNA HS Kit on Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA). Quality and peak size was determined with the D1000 ScreenTape on Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA). Final libraries were diluted to 4 nM and libraries of different indices were pooled for sequencing together in equimolar amounts. Each pooled library at a final concentration of about 2.1 pM was clustered using Illumina’s NextSeq 550 FlowCell and sequenced using NextSeq 550 high output kit (Illumina, Inc., San Diego, CA). Single reads with 75bp (RRBS) and 100bp (RNA-seq) were generated.

RNA was extracted following the Direct-zol RNA MicroPrep kit (Zymo Research) manufacturer’s instructions. RNA was treated with DNAse I (Ambion) and enriched for mRNA using Dynabeads mRNA DIRECT Purification Kit (Thermo Fisher). cDNA was generated using a custom scaled-down modification of the SMART-seq protocol [36 (link)]. cDNA was synthesized from RNA input using Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific). It was then amplified using Kapa HiFi 2X Ready Mix (Kapa Biosystems) and cleaned using 0.9X Ampure beads (Beckman Coulter). Finally, cleaned cDNA samples were tagmented and indexed using the Nextera XT DNA Library Prep Kit (Illumina). Library size and tagmentation were confirmed using the TapeStation HS D1000 kit (Agilent). Libraries were pooled in equal molarity and sequenced with the NextSeq 550 High-Output kit (Illumina) with paired-end 75 bp reads.