Agilent 2100 bioanalyzer on

To obtain the artificial single-source degraded DNA, the DNA with the highest heterozygosity from the 32 samples was incubated at 98°C for 35, 40, and 45 min in the Eppendorf 6331 Nexus Gradient Flexlid Thermal Cycler (Eppendorf, Hamburg, Germany). In addition, the standard DNA M308 was incubated in a PCR system at 98°C for 120, 160, and 170 min.
To obtain artificially mixed and degraded DNA, the two DNA samples with the highest degree of heterozygosity and homozygosity from the 32 samples were mixed and then subjected to a series of dilutions (1:1, 1:10, 1:20, 1:50, 1:100, 1:500, and 1:1,000) and incubated at 98°C for 35, 40, and 45 min.
The degree of DNA degradation after incubation was determined using a High Sensitivity DNA Kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States) and the AGCU EX22 Kit (Applied ScienTech, Jiangsu, China) on an ABI 3500 Genetic Analyzer (Applied Biosystems), according to the manufacturers’ instructions. The results were analyzed using the GeneMapper ID-X v1.2 software (Applied Biosystems).

For dsRNA and ribo-depleted totRNA, random cDNA was synthesized using ProtoScript II Reverse Transcriptase and random octamer primers (8N). A denaturation step of 99°C for 2 min for the dsRNA and 65°C for 5 min for the ribo-depleted totRNA. ds-cDNA was synthesized using NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs). Libraries were prepared using Nextera DNA Library Prep Kit (Illumina) following the manufacturer protocol. The quantification was done using Qubit dsDNA HS Assay Kit (Life Technologies) and quality analysis was done using High Sensitivity DNA Chips on Agilent 2100 Bioanalyzer (Agilent Technologies) following the manufacturers’ protocols. Subsequently, the libraries were sequenced on a MiSeq Illumina platform v.3 pair-end reads (2x301) at DSMZ (Braunschweig, Germany). For the sRNA, libraries were prepared from sRNA extracted using TruSeq small RNA kit (Illumina) at Fasteris Life Sciences SA (Plan-les-Ouates, Switzerland) and sequenced on a NextSeq Illumina platform single-end reads (1x50). For the RCA products, the library was also prepared using Nextera DNA Library Prep Kit and run on a NextSeq Illumina platform (2x151) at DSMZ (Braunschweig, Germany).

Ten nanograms of DNA was used for multiplex PCR amplification. Libraries were constructed using the Ion AmpliSeq Library Kit v2.0 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The quality of the obtained libraries was evaluated using Agilent 2100 Bioanalyzer on-chip electrophoresis (Agilent Technologies, Palo Alto, CA, USA). Emulsion PCR was performed with the OneTouch DL or OneTouch 2 system (Thermo Fisher Scientific). Sequencing was run on the Ion Torrent Personal Genome Machine (Thermo Fisher Scientific), loaded with a 316 or 318v2 chip as per the manufacturer’s protocol. Data analysis, including alignment to the hg19 human reference genome as well as variant calling and filtering, was completed using Torrent Suite Software v3.6 (Thermo Fisher Scientific). Filtered variants were annotated using Ion Reporter software v4.4 (Thermo Fisher Scientific).