Biology subjects

Schimke, K. D.

Publications and source records attributed to Schimke, K. D..

3 recordsLinked to original sources

bioRxiv2025

Sequencing and assembling complete plasmids on Oxford Nanopore Technology Sequencers using R2C2 and Chopper

Plasmids are ubiquitous tools in molecular biology which are used for a large variety of experiments within academic and commercial labs. Both new and old plasmids have to undergo sequencing-based analysis to determine whether or not they are functional, i.e. contain the correct insert in the correct backbone. While traditional Sanger sequencing based analysis was most often limited to the inserts, new high-throughput sequencing based methods and services can now provide the complete sequence of a plasmid. Currently available methods and services vary in throughput and cost. Here, we adapted the Oxford Nanopore Technologies-based R2C2 sequencing method to - rapidly and at low cost - sequence complete plasmids, either individually or in a pool. We also developed an analysis pipeline, Chopper, that produces full-length plasmid sequences. We tested our workflow with commonly used plasmids we ordered from Addgene and produced highly accurate sequences for each plasmid from both their individual and pooled sequencing runs.

bioinformatics↗

bioRxiv2022

Identifying and quantifying isoforms from accurate full-length transcriptome sequencing reads with Mandalorion

The Mandalorion tool, which we have continuously developed over the last 5 years, identifies and quantifies high-confidence isoforms from accurate full-length transcriptome sequencing reads produced by methods like PacBio Iso-Seq and ONT-based R2C2. In this manuscript, we introduce and benchmark Mandalorion v4 which further improves upon the already strong performance of Mandalorion v3.6 used in the LRGASP consortium challenge. By processing real and simulated accurate full-length transcriptome sequencing data sets, we show three main features of Mandalorion: First, Mandalorion-based isoform identification has very high Precision and maintains high Recall even when used in the absence of any genome annotation. Second, isoform read counts as quantified by Mandalorion show high correlation with simulated read counts. Third, isoforms identified by Mandalorion closely reflect the full-length transcriptome sequencing data sets they are based on.

bioinformatics↗

bioRxiv2021

Illumina But With Nanopore: Sequencing Illumina libraries at high accuracy on the ONT MinION using R2C2

High-throughput short-read sequencing has taken on a central role in research and diagnostics. Hundreds of different assays exist today to take advantage of Illumina short-read sequencers, the predominant short-read sequencing technology available today. Although other short read sequencing technologies exist, the ubiquity of Illumina sequencers in sequencing core facilities, and the high capital costs of these technologies have limited their adoption. Among a new generation of sequencing technologies, Oxford Nanopore Technologies (ONT) holds a unique position because the ONT MinION, an error-prone long-read sequencer, is associated with little to no capital cost. Here we show that we can make short-read Illumina libraries compatible with the ONT MinION by using the R2C2 method to circularize and amplify the short library molecules. This results in longer DNA molecules containing tandem repeats of the original short library molecules. This longer DNA is ideally suited for the ONT MinION, and after sequencing, the tandem repeats in the resulting raw reads can be converted into high-accuracy consensus reads with similar error rates to that of the Illumina MiSeq. We highlight this capability by producing and benchmarking RNA-seq, ChIP-seq, as well as regular and target-enriched Tn5 libraries. We also explore the use of this approach for rapid evaluation of sequencing library metrics by implementing a real-time analysis workflow.

genomics↗