Journal: bioRxiv
Article Title: ConSeqUMI, an error-free nanopore sequencing pipeline to identify and extract individual nucleic acid molecules from heterogeneous samples
doi: 10.1101/2025.04.03.647077
Figure Lengend Snippet: ( A ) Chimeric read experiment schematic. UMI tags were appended to flanking regions of the degenerate barcode of a pool barcoded plasmids using two cycles of PCR with universal primer binding sites. Following bead cleanup, amplicons were further amplified with additional PCR cycles using appended constant regions as primer binding sites. Amplicons were prepared for nanopore sequencing using v10 chemistry native barcoding and sequenced on a MinION sequencer using an R9 flow cell. B ) Cluster sizes in reads of non-chimeric and chimeric UMI pairings for 1.5 × 10 6 (top panel) or 1.5 × 10 5 (bottom panel) input molecules following ConSeqUMI processing. Clusters containing chimeric UMIs are displayed in blue. Inset box plots summarize the data of the quantile plots. C ) Isolation of two transgenes from heterogeneous pools via PCR using UMIs as primer binding sites. Abridged cluster consensus sequences for each ConSeqUMI algorithm are shown below the template sequence region containing degenerate nucleotides, with the constant backbone regions in gray. Chromatograms display the results of Sanger sequencing of the resulting amplicons. D ) Plasmid pool experiment schematic. UMI tags were appended to variable regions of a pool of 14 pcDNA3.1-based plasmids using two cycles of PCR with universal primer binding sites on the pcDNA3.1 backbone. Following bead cleanup, amplicons were further amplified with additional PCR cycles using appended constant regions as primer binding sites. Amplicons were prepared for sequencing using v14 chemistry native barcoding and sequenced on a MinION sequencer using an R10 flow cell. E ) Replicate UMI-labeled plasmid pool samples were sequenced and clustered using ConSeqUMI. Primer-based UMI addition showed consistent cluster identity proportions across replicates per input amount.
Article Snippet: MinION Mk1B Sequencing Device (MIN-101B), Ligation Sequencing Kit (SQK-LSK109) with the Adapter Mix II Expansion (EXP-AMII001), Spot-ON Flow Cell, R9 Version (FLO-MIN106D), MinION Flow Cell, R10 Version (FLO-MIN114), Flongle Flow Cell, R10 Version (FLO-FLG114), Flongle Sequencing Expansion (EXP-FSE002), and the Native Barcoding Kit 96 v14 (SQK-NBD114.96) were obtained from Oxford Nanopore Technologies (Oxford, United Kingdom). dPTP (6H,8H-3,4-Dihydro-pyrimido(4,5-c)(1,2)oxazin-7-one-8-β-D-2’-deoxy-ribofuranoside-5’-triphosphate, Sodium salt) (NU-1119S) was obtained from Jena Bioscience (Jena, Germany).
Techniques: Binding Assay, Amplification, Nanopore Sequencing, Isolation, Sequencing, Plasmid Preparation, Labeling
