spotPCR: A Rapid and Efficient Approach for Indexing Individual Template Molecules using Unique Molecular Identifiers.

Low-frequency mutations provide valuable insights in various fields, including drug resistance identification, cancer and infectious disease research. One promising strategy to enhance the sensitivity and specificity of mutation detection is the incorporation of unique molecular identifiers (UMIs) during polymerase chain reaction (PCR) amplification and before deep sequencing. However, conventional methods for UMI incorporation often necessitate multiple labor-intensive steps.

stilPCR increases the effective sequencing length of Illumina targeted next-generation sequencing.

Identifying pathogens, resistance-conferring mutations, and strain types through targeted amplicon sequencing is an important tool. However, due to the limitations of short read sequencing, many applications require the division of limited clinical samples. Here, we present stilPCR (single-tube Illumina long read PCR), which allows the generation of hemi-nested amplicons in a single tube, with Illumina indexes and adapters, effectively increasing the Illumina read length without increasing the input requirements of reagents or sample.

Turbolysis: A low-cost, small footprint alternative to commercial bead beaters for cell lysis.

turboLysis is a novel mechanical cell lysis device that utilizes small beads to efficiently lyse tough cells like Mycobacterium, Saccharomyces, and Arabidopsis. We compared turboLysis to bead beating using the BeadBug 6 for several concentrations of roughly correlated to the bacterial load commonly seen in patient samples. turboLysis performed similarly to the BeadBug at low bacterial concentrations and outperformed it at high concentrations above 2x10 CFU/ml (p < 0.

primerJinn: a tool for rationally designing multiplex PCR primer sets for amplicon sequencing and performing in silico PCR.

Background: Multiplex PCR amplifies numerous targets in a single tube reaction and is essential in molecular biology and clinical diagnostics. One of its most important applications is in the targeted sequencing of pathogens. Despite this importance, few tools are available for designing multiplex primers.

selSeq: A method for the enrichment of non-polyadenylated RNAs including enhancer and long non-coding RNAs for sequencing.

Non-polyadenylated RNA includes a large subset of crucial regulators of RNA expression and constitutes a substantial portion of the transcriptome, playing essential roles in gene regulation. For example, enhancer RNAs are long non-coding RNAs that perform enhancer-like functions, are bi-directionally transcribed, and usually lack polyA tails. This paper presents a novel method, selSeq, that selectively removes mRNA and pre-mRNA from samples enabling the selective sequencing of crucial regulatory elements, including non-polyadenylated RNAs such as long non-coding RNA, enhancer RNA, and non-canonical mRNA.

A simple, single-tube overlapping amplicon-targeted Illumina sequencing assay.

Targeted amplicon sequencing to identify pathogens, resistance-conferring mutations, and strain types is an important tool in diagnosing and treating infections. However, due to the short read limitations of Illumina sequencing, many applications require the splitting of limited clinical samples between two reactions. Here, we outline hairpin Illumina single-tube sequencing PCR (hissPCR) which allows for the generation of overlapping amplicons containing Illumina indexes and adapters in a single tube, effectively extending the Illumina read length while maintaining reagent and sample input requirements.

primerJinn - a tool for rationally designing multiplex PCR primer sets and in silico PCR.

Background: Multiplex PCR amplifies numerous targets in a single tube reaction and is essential in molecular biology and clinical diagnostics. One of its most important applications is in the targeted sequencing of pathogens. Despite this importance, few tools are available for designing multiplex primers.

Circularization of for Genotypic Bedaquiline Resistance Testing of Mycobacterium tuberculosis.

Circular DNA offers benefits over linear DNA in diagnostic and field assays, but currently, circular DNA generation is lengthy, inefficient, highly dependent on the length and sequence of DNA, and can result in unwanted chimeras. We present streamlined methods for generating PCR-targeted circular DNA from a 700 bp amplicon of , the high GC content (65%) gene implicated in Mycobacterium tuberculosis bedaquiline resistance, and demonstrate that these methods work as desired. We employ self-circularization with and without splints, a Gibson cloning-based approach, and novel 2 novel methods for generating pseudocircular DNA.

Outcomes, infectiousness, and transmission dynamics of patients with extensively drug-resistant tuberculosis and home-discharged patients with programmatically incurable tuberculosis: a prospective cohort study.

Background: The emergence of programmatically incurable tuberculosis threatens to destabilise control efforts. The aim of this study was to collect prospective patient-level data to inform treatment and containment strategies.

Methods: In a prospective cohort study, 273 South African patients with extensively drug-resistant tuberculosis, or resistance beyond extensively drug-resistant tuberculosis, were followed up over a period of 6 years.