Mastering Next Gen Sequencing: Elevate Your Workflow

Illumina® RNA Prep with Enrichment on Eppendorf’s epMotion® 5075t NGS Solution SHORT PROTOCOL No. 51 Next Generation Sequencing (NGS) is a powerful tool that is used in various scientifi c fi elds and applications. One, that is of special interest is the identifi cation, characterization and continuous surveillance of known and emerging viruses, like SARS-CoV-2, Infl uenza and others. For that purpose, Illumina’s RNA prep with Enrichment kit can perfectly be combined with the Respiratory Virus Oligos Panel (RVOP). It uses a target enrichment through hybrid–capture resequencing approach which allows highly sensitive detection without requiring the high read depth needed for shotgun metagenomics sequencing. Furthermore, it provides an eff ective way to detect a broad range of viruses without specifi cally knowing the infectious agent fi rst, and obtains reliable sequence data, enabling applications such as variant analysis for viral surveillance and evolution. The target enrichment is facilitated by capturing genomic regions of interest by hybridization to target-specifi c biotinylated probes. The collection of probes used in this protocol make up the Respiratory Virus Oligo Panel v2, targeting numerous viruses, relevant for the monitoring of respiratory disease status. Preparing samples for NGS is a multi-step process that may stretch over several days and requires high precision. Automating this procedure on the Eppendorf epMotion 5075t NGS solution (Fig. 1) can increase the throughput and reproducibility while at the same time minimizing the potential for human error. Furthermore, it reduces the required hands-on time by up to 80%, freeing up time for other activities. The method described here, was tested in conjunction with Illumina. Protocol structure Instrument configuration epMotion 5075t NGS solution No. of sub-methods 8 No. of samples 24-96 (pre-enrichment) 8-32 (post-enrichment) Total processing time* 5 h (24 samples) – 9 h (96 samples) Hands-on time approx. 45 min Pooling approach 3-plex; pool by volume (2,5 µL/sample) *w/o considering off-deck incubation times Figure 1: epMotion 5075t with MultiCon control panel. SHORT PROTOCOL I Page 2 Qualification data > The epMotion workflow is exemplarily shown for a sample input of 25 ng Universal Human Reference RNA (UHR, Agilent®, #740000 ) with spiked in SARS-CoV-2 control RNA 2 (TWIST™ Bioscience, #MN908947.3) in copy numbers of 0, 10, 100 and 1,000 per sample. > QC was performed using the Qubit™ dsDNA HS Assay (Invitrogen™ #Q32851) ahead of method 4 (prior to pooling of pre-enriched libraries). Library concentrations showed an average of 146 ng/µL. To stay within Illumina’s recommendation sample concentrations were adjusted Sequencing and data analysis Sequencing was performed on Illumina’s NextSeq® 550 with a starting concentration of 4 nM. For data analysis the DRAGEN® Pathogen Detection and DRAGEN Metagenomics Pipeline was used. Data analysis is based on k-mers and alignment steps, including protein encoding transcript level detection of viruses, Summary Illumina’s RNA Prep with Enrichment enables researchers to identify and characterize known and emerging viruses, which is central to improving public health. Seeing the importance thereof, Eppendorf has established a qualified method for the epMotion 5075t NGS solution, producing high quality sequencing libraries that are comparable to libraries from manual processing, while increasing productivity by freeing up time for other activities. before proceeding with method 4 (pooling) to not exceed the max. input of 600 ng DNA per pool using a 3-plex pooling approach. > The final libraries were analyzed again using the Qubit™ dsDNA HS Assay, showing concentrations of 1.39 ng/µL on average. Additional analysis was carried out with the Bioanalyzer® High Sensitivity DNA Analysis Kit (Agilent®, #5067-4626) showing an evenly distributed peak around an average fragment size of 357 bp (Fig. 2A). which increases the ability to identify novel and highly divergent viruses. The analysis showed classified reads for SARS-CoV-2 at concentrations as low as 10 copies per sample (Fig. 2C). Nearly full genome coverage was achieved at 1,000 copies with minimal coverage being observed with as low as 10 copies (Fig. 2B). SHORT PROTOCOL I Page 3 A B C Figure 2: Collected data of Illumina’s RNA Prep with Enrichment performance on an Eppendorf epMotion 5075t NGS Solution. A: Overlay of final libraries that were analyzed with the Bioanalyzer high sensitivity DNA Assay. The electro- pherogram shows evenly distributed peaks around an average fragment size of 357 bp. B: SARS-CoV-2 Genome coverages at different viral copy numbers spiked into the sample – as determined by sequencing on a NextSeq 550. C: Number of SARS-CoV-2 reads