D5000 screentape

We visualized cfDNA profiles on High Sensitivity D5000 ScreenTapes and a TapeStation 4200 system (Agilent Technologies, Santa Clara, CA, USA). The equivalent of 0.08 mL urine or plasma was visualized for each specimen.

Oxford Nanopore sequencing was used to measure the accuracy and purity of plasmid template preparations. Ligation Sequencing libraries (SQK-LSK109) were prepared from linearised plasmid templates (described above) and were labelled with PCR-Free Native Barcodes (EXP-NBD104). Libraries were prepared according to manufacturer’s instructions (Oxford Nanopore Technologies), with the exception that 2 µg of template was used as an input rather than the recommended 1 µg. The resulting libraries were quantified on a Qubit instrument (Invitrogen) with the dsDNA HS kit and qualitative analysis of fragment length distribution was completed with D5000 ScreenTapes (Agilent Technologies, USA). The results of the quantitative and qualitative analyses were used to calculate the required library concentrations for pooling and loading. Barcoded libraries were sequenced on either R9.4.1 (FLO-MIN106D) or Flongle Flow Cells, with High Accuracy live base-calling enabled (Guppy v5.1.13 and MinKNOW Core 4.5.4). All nanopore reads with quality scores >9 were used to form a concatenated FASTQ file and proceeded to further analysis.

For sequencing of validation samples, Illumina’s next-generation sequencing methodology^{69 (link)} was used. In detail, genomic DNA was quality-checked and quantified using the 4200 TapeStation instruments in combination with the Genomic DNA ScreenTape (both Agilent Technologies). Libraries were prepared from 100 ng of input material using Ovation RRBS Methyl-Seq with TrueMethyl oxBS (Tecan, Cat.no: 0553-32). In detail, only the bisulfite part of the protocol was followed, oxidation of DNA was not done, and samples were treated as MOCK oxBS samples according to the manufacturer’s instruction. Quantification and quality checks of libraries were done using the 4200 TapeStation and D5000 ScreenTapes instruments (both Agilent Technologies). Libraries were pooled and sequenced on a NovaSeq 6000 (Illumina) using S1 100-cycle reagents (Illumina, Cat.no: 20028319). The system was running in 101 cycle/single-end/standard loading workflow mode. Sequence information was converted to FASTQ format using bcl2fastq v2.20.0.422.

cDNA was prepared using a SMART-Seq v4 Ultra Low Input RNA kit for sequencing (Takara Bio, 634898). The cDNA quality was examined on an Agilent TapeStation system using a High-Sensitivity D5000 ScreenTape (Agilent, 5067-5592). One nanogram of cDNA was used for library preparation using a Nextera XT DNA library preparation kit (Illumina, FC-131-1024 and FC-131-1096). The yield and quality of the amplified libraries were analyzed using Qubit (Thermo Fisher) and the Agilent TapeStation. The indexed cDNA libraries were normalized and combined, and the pools were sequenced on an Illumina NextSeq 550 for a 75-cycle v2 sequencing run generating 75-base pair single-end reads.

Up to 50,000 cells per sample were tagmented with Nextera Tn5 transposase using the Illumina Nextera kit and purified with a Qiagen MinElute reaction kit per methods modified from Buenrostro et al. (2013) (link). The DNA was then PCR amplified to add indexing primers in nine cycles. SPRI AMPure beads enriched for fragments under ∼600 bp. The library was then amplified again with the nine-cycle protocol, followed by cleanup with SPRI AMPure beads. The DNA library was quantified with a Qubit DNA High Sensitivity assay and analyzed for quality and size distribution on an Agilent 2200 TapeStation with a High Sensitivity D5000 ScreenTape. Samples were pooled at a 10 nM final concentration. Sequencing was run with an Illumina HiSeq 2500 system with 2 × 50 bp read length by the Washington University in St Louis School of Medicine GTAC.

Amplified cDNA products were then fragmented for next-generation sequencing using the Nextera XT DNA Sample Prep Kit (Illumina). The resultant sequencing pool was subjected to TapeStation 4200 electrophoresis using High Sensitivity D5000 ScreenTape (Agilent Technology). Next-generation sequencing was then performed with HiSeq 2500 (Illumina; performed by Macrogen Corp., Japan). Pair-end reads of 101 nucleotides were sequenced.