Nextseq 500 mid output v2 kit

Whole genome sequencing of all the study isolates was done using previously described methods (Pintara et al., 2018 ).
QIAamp DSP DNA mini kit (Qiagen, Germany) was used to extract DNA from the isolates using the QIAsymphony SP, following the Tissue HC 200 V DSP protocol. Quantitation of extracted DNA was done on a plate reader using the Quant-IT kit (Thermo Fisher Scientific, United States).
The Nextera XT DNA library preparation kit (Illumina, United States) was used for library preparation of the DNA samples. The DNA samples were then sequenced on the NextSeq500 (Illumina, United States) using the NextSeq 500 Mid Output V2 kit (Illumina, United States).
Trimmomatic v0.36 was used to trim sequence reads from the sequenced isolates and quality checked by FastQC v0.11.5 (Andrews, 2014 ; Bolger et al., 2014 (link)). Trimmed reads were then de novo assembled using the SPAdes assembler version 3.9.1 (Bankevich et al., 2012 (link)) and assemblies annotated using Prokka (Overbeek et al., 2014 (link)).

Genetic sequencing was performed in all the patients with suspected DCM. Genomic DNA was extracted from patient’s whole blood using Chemagic DNA blood kit (PerkinElmer, Inc. Waltham, Massachusetts, USA) following the manufacturer’s protocol. Quality and quantity of the extracted DNA were assessed by Labchip Ds (PerkinElmer, Inc), an automated ultraviolet-visible spectrophotometer. Purified DNA was targeted enriched for genes related to DCM using TruSight Cardio kit (Illumina, San Diego, California, USA) according to the manufacturer’s protocol. Pooled libraries were sequenced using Illumina MiSeq (v2 kit) or NextSeq 500 (Mid Output v2 kit) benchtop sequencers using paired-end, 150 bp reads. Raw sequencing data were demultiplexed, trimmed and mapped to UCSC GRCh37/hg19 reference genome as previously described [11 (link)]. Variants were called using GATKv3.3 HaplotypeCaller and UnifiedGenotyper annotated using CardioClassifier, a semi-automatic classification on inherited cardiac conditions defined according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology [12 (link)]. The pathogenicity of the variants for each patient was further confirmed by a cardiologist who is an expert in genetics and DCM (SAC). He was blinded to the imaging and other clinical data.

Whole genome sequencing was performed on the isolates listed in Supplementary Table S1 using the procedure described in our previous studies [14 (link), 15 (link)]. Briefly, DNA was prepared for sequencing using the Nextera XT kit (Illumina, San Diego, USA) and sequenced on the NextSeq500 using the NextSeq 500 Mid Output v2 kit (300 cycles) (Illumina), according to the manufacturer’s instructions. Sequence reads for the C. diphtheriae isolates were trimmed with Trimmomatic v0.36 [16 (link)] and quality checked by FastQC v0.11.5 and MultiQC v1.1 [17 (link)]. Sequences were de novo assembled into contigs using the SPAdes assembler v3.12.0 [18 (link)]. Sequence files have been submitted to the European Nucleotide Archive under project accession number PRJEB58646.

Allantoic fluids (strains 23/2013 and 38/2013, P1 and P3) were clarified by
centrifugation at 16,000 x g for 3 min at4 ºC, filtered through
0.45 μm syringe filters, and treated with DNase-free RNase. Total RNA was then
extracted with Trizol Reagent (Life Technologies) and RNeasy Mini kit (Qiagen),
and used with Superscript III and Klenow exo-DNA polymerase (Life Technologies)
to obtain random ds-cDNAs.
Libraries and sequencing kits were Nextera XT Index and Nextera XT DNA
(Illumina), and reads were obtained with a NextSeq500 (Illumina) sequencer using
the NextSeq500 Mid output v2 kit (2 x 150 bp). Read quality was assessed with
FASTQC , and full genomes were
assembled with CLC Genomics Workbench v. 11.0.1 (Qiagen), with the
reference-mapping approach.