Kapa library quantification kits for platforms

Viral RNA samples were obtained from the first passage of Vero cell isolates from urine of patient 1 and saliva of patient 6. Double-stranded cDNA libraries were prepared using the TruSeq Stranded mRNA LT Sample Preparation Kit (Illumina, San Diego, CA, USA). Briefly, the polyA containing mRNA purification step was not performed and the protocol was started with 25–35 ng of RNA in 5 ul of molecular biology grade water to which were added 13 ul of Fragment, Prime, Finish Mix. The remaining steps of the protocol were carried out without any modifications. Library quality control was performed using the 2100 Bioanalyzer System with the Agilent DNA 1000 Kit (Agilent, Santa Clara, CA, USA). The libraries were individually quantified via qPCR using a KAPA Library Quantification Kits for Illumina platforms (KAPA Biosystems, Wilmington, MA, USA). The libraries were pooled together in equimolar quantities and sequenced. Paired-end reads (2 × 75 bp) were obtained using a MiSeq Reagent Kits v3 (150-cycles) in a MiSeq sequencing system (Illumina).

Exome enrichment was accomplished with the NimbleGen SeqCap EZ Exome+UTR (Roche NimbleGen, version 2) that targets 64 Mb of coding exons and miRNA regions plus 32 Mb untranslated regions (UTRs) for solution-based capture following the manufacturer’s protocol. Library preparation was performed with 200 ng of genomic DNA using KAPA HyperPlus library kit (Roche, KK8514) using adaptors compatible with Illumina sequencer on the Hamilton STAR automated platform. We performed amplification, pooling, hybridization, washing, and elution according to the manufacturer’s instructions. We assessed the libraries for quality with a high sensitivity DNA ScreenTape assay on the 2200 TapeStation System (Agilent) and quantity with KAPA Library Quantification Kits for Illumina platforms (Kapa Biosystems). The libraries were diluted to 2 nM and clustered using an Illumina cBot with a HiSeq 3000/4000 paired-end cluster kit on a patterned flow cell and a HiSeq 3000/4000 SBS kit (300 cycles, Illumina v2.5 reagents) on the HiSeq 4000 sequencing platform.

We captured in total 3801 diploid and 4939 trisomic single-cell fibroblasts from the monozygotic twin pair using the Chromium System powered by GemCode Technology (10x Genomics). Single-cell RNA-seq libraries were generated using the Chromium Single Cell 3' Reagent Kit version 2 (10x Genomics) according to the manufacturer’s instructions. Briefly, the concentration of trypsin dissociated fibroblasts was set to 1500 cells/µl of culture medium (Dulbecco’s Modified Eagle Medium (DMEM), 10% FBS) and 5000 individualized cells were flown per channel following the recommendation of the manufacturer. All libraries were quantified by Qubit (Invitrogen) and by quantitative real-time PCR using the PCR-based KAPA Library Quantification Kits for Illumina platforms (Kapabiosystems). Size profiles of the pre-amplified cDNA and sequencing libraries were assessed using a 2100 BioAnalyzer (Agilent) with a High Sensitivity DNA chip kit (Agilent). Barcoded libraries were sequenced with an HiSeq 4000 (Illumina) as paired-end 100 bp reads as recommended by 10x Genomics. The proprietary software CellRanger (10x Genomics) with default parameters was used in order to demultiplex the samples and quantify the abundance of mRNA molecules (UMI - Unique Molecular Identifier). Processed data were analyzed using custom R scripts.

The 12 transcriptome libraries were prepared from 500 ng of total RNA using the TruSeq Stranded Total RNA Sample Preparation Kit with Ribo-Zero Gold (Illumina). Additionally, the 12 small RNA libraries were prepared from 1 μg from the same total RNA of transcriptome analysis, using the TruSeq Small RNA Library Prep Kit (Illumina). All the steps of the protocols were carried out without any modifications, with the exception of the cDNA construction purification step on the small RNA protocol, which was performed using 3% Agarose Gel Cassette for targets between 100 bp − 250 bp in a BluePippin system (Sage Science). The quality control of each library was performed using the 2100 Bioanalyzer System with the Agilent High Sensitivity DNA Kit (Agilent). The libraries were quantified via qPCR using a KAPA Library Quantification Kits for Illumina platforms (KAPA Biosystems). The Libraries were sequenced on a NextSeq. 500 sequencing system (Illumina), for the RNA-seq paired-end reads (2 × 75 bp) were obtained using NextSeq. 500/550 High Output v2 kit (150 cycles) and for the small RNA, single-end reads (1 × 50 bp) were obtained using a NextSeq. 500/550 High Output v2 kit (75 cycles).

NGS libraries were prepared from cfDNA according to the manufacturer’s instructions (protocol based on QIAseq Targeted DNA Panel Handbook, R2; May 2017, Qiagen, Hilden, Germany) except for the DNA fragmentation. End-repair and Poly(A) tailing were followed by Illumina NGS adapter (Illumina, San Diego, CA, USA) ligation to cfDNA containing a sample index and a unique molecular identifier sequence (UMI). After UMI assignment, target enrichment of ligated cfDNA was performed by PCR using target specific DEEPGEN^TM primers. Library concentrations were determined with KAPA Library Quantification Kits for Illumina platforms (Roche Holding AG, Basel, Switzerland). Libraries were prepared using NovaSeq Reagent Kits (Illumina, San Diego, CA, USA) and sequenced with a 300-cycle S4 kit on a NovaSeq 6000 (Illumina, San Diego, CA, USA) with a mean raw sequencing depth of ~200,000×. 3062 genomic variants were targeted. All steps were carried out according to the manufacturer’s instructions.