Single Molecule Molecular Inversion Probes for High Throughput Germline Screenings in Dystonia

AbstractTemperature sensitive (TS) alleles are important tools for the genetic and functional analysis of essential genes in many model organisms. While isolating TS alleles is not difficult, determining the TS-conferring mutation can be problematic. Even with whole-genome sequencing (WGS) data there is a paucity of predictive methods for identifying TS alleles from DNA sequence alone. We assembled 173 TS lethal mutants of Caenorhabditis elegans and used WGS to identify several hundred mutations per strain. We leveraged single molecule molecular inversion probes (MIPs) to sequence variant sites at high depth in the cross-progeny of TS mutants and a mapping strain with identified sequence variants but no apparent phenotypic differences from the reference N2 strain. By sampling for variants at ~1Mb intervals across the genome we genetically mapped mutant alleles at a resolution comparable to current standards in a process we call MIP-MAP. The MIP-MAP protocol, however, permits high-throughput sequencing of multiple TS mutation mapping libraries at less than 200K reads per library. Using MIP-MAP on a subset of TS mutants, via a competitive selection assay and standard recombinant mutant selection, we defined TS-associated intervals of 3Mb or less. Our results suggest this collection of strains contains a diverse library of TS alleles for genes involved in development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes. The MIPs protocol should allow high-throughput tracking of genetic variants in any mixed population.

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Highly Sensitive Detection of Paraquat with Pillar[5]arene as an aptamer in α-Hemolysin Nanopore

Materials Chemistry Frontiers ◽

10.1039/d1qm00875g ◽

2021 ◽

Author(s):

Xiaojia Jiang ◽

Mingsong Zang ◽

Fei Li ◽

Chunxi Hou ◽

Quan Luo ◽

...

Keyword(s):

Real Time ◽

High Throughput ◽

Single Molecule ◽

Single Molecule Detection ◽

Sensitive Detection ◽

High Throughput Analysis ◽

Throughput Analysis ◽

The Real ◽

Highly Sensitive ◽

Highly Sensitive Detection

Biological nanopore-based techniques have attracted more and more attention recently in the field of single-molecule detection, because they allow the real-time, sensitive, high-throughput analysis. Herein, we report an engineered biological...

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Parallel, linear, and subnanometric 3D tracking of microparticles with Stereo Darkfield Interferometry

Science Advances ◽

10.1126/sciadv.abe3902 ◽

2021 ◽

Vol 7 (6) ◽

pp. eabe3902

Author(s):

Martin Rieu ◽

Thibault Vieille ◽

Gaël Radou ◽

Raphaël Jeanneret ◽

Nadia Ruiz-Gutierrez ◽

...

Keyword(s):

High Precision ◽

High Throughput ◽

Single Molecule ◽

Particle Tracking ◽

Focal Plane ◽

Tracking System ◽

Three Dimensional ◽

3D Tracking ◽

Single Molecule Force Spectroscopy ◽

A New Technique

While crucial for force spectroscopists and microbiologists, three-dimensional (3D) particle tracking suffers from either poor precision, complex calibration, or the need of expensive hardware, preventing its massive adoption. We introduce a new technique, based on a simple piece of cardboard inserted in the objective focal plane, that enables simple 3D tracking of dilute microparticles while offering subnanometer frame-to-frame precision in all directions. Its linearity alleviates calibration procedures, while the interferometric pattern enhances precision. We illustrate its utility in single-molecule force spectroscopy and single-algae motility analysis. As with any technique based on back focal plane engineering, it may be directly embedded in a commercial objective, providing a means to convert any preexisting optical setup in a 3D tracking system. Thanks to its precision, its simplicity, and its versatility, we envision that the technique has the potential to enhance the spreading of high-precision and high-throughput 3D tracking.

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Single molecule high-throughput footprinting of small and large DNA ligands

Nature Communications ◽

10.1038/s41467-017-00379-w ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 18

Author(s):

Maria Manosas ◽

Joan Camunas-Soler ◽

Vincent Croquette ◽

Felix Ritort

Keyword(s):

High Throughput ◽

Single Molecule

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Identification of community-consensus clinically relevant variants and development of single molecule molecular inversion probes using the CIViC database

10.1101/479394 ◽

2018 ◽

Author(s):

Erica K. Barnell ◽

Adam Waalkes ◽

Kelsi Penewit ◽

Katie M. Campbell ◽

Zachary L. Skidmore ◽

...

Keyword(s):

Single Molecule ◽

Pearson Correlation ◽

Genomic Region ◽

Variant Annotation ◽

Design Paradigm ◽

Molecular Inversion Probes ◽

Sequencing Platforms ◽

Tumor Types ◽

Molecular Inversion Probe ◽

Actionable Variants

AbstractClinical targeted sequencing panels are important for identifying actionable variants for cancer patients, however, there are currently no strategies to create impartial and rationally-designed panels to accommodate rapidly growing knowledge within the field. Here we use the Clinical Interpretations of Variants in Cancer database (CIViC) in conjunction with single-molecule molecular inversion probe (smMIP) capture to identify and design probes targeting clinically relevant variants in cancer. In total, 2,027 smMIPs were designed to target 111 eligible CIViC variants. The total genomic region covered by the CIViC smMIPs reagent was 61.5 kb. When compared to existing genome or exome sequencing results (n = 27 cancer samples from 5 tumor types), CIViC smMIP sequencing demonstrated a 95% sensitivity for variant detection (n = 61/64 variants). Variant allele frequency for variants identified on both sequencing platforms were highly concordant (Pearson correlation = 0.885; n = 61 variants). Moreover, for individuals with paired tumor/normal samples (n = 12), 182 clinically relevant variants missed by original sequencing were discovered by CIViC smMIPs sequencing. This design paradigm demonstrates the utility of an open-sourced database built on attendant community contributions for each variant with peer-reviewed interpretations. Use of a public repository for variant identification, probe development, and variant annotation could provide a transparent approach to build a dynamic next-generation sequencing–based oncology panel.

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ProtSeq: towards high-throughput, single-molecule protein sequencing via amino acid conversion into DNA barcodes

iScience ◽

10.1016/j.isci.2021.103586 ◽

2021 ◽

pp. 103586

Author(s):

Jessica M. Hong ◽

Michael Gibbons ◽

Ali Bashir ◽

Diana Wu ◽

Shirley Shao ◽

...

Keyword(s):

Amino Acid ◽

High Throughput ◽

Single Molecule ◽

Protein Sequencing ◽

Dna Barcodes

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A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq

PeerJ ◽

10.7717/peerj.2492 ◽

2016 ◽

Vol 4 ◽

pp. e2492 ◽

Cited By ~ 29

Author(s):

Catherine M. Burke ◽

Aaron E. Darling

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Single Molecule ◽

Illumina Miseq ◽

Full Length ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Bacterial Taxonomy

BackgroundThe bacterial 16S rRNA gene has historically been used in defining bacterial taxonomy and phylogeny. However, there are currently no high-throughput methods to sequence full-length 16S rRNA genes present in a sample with precision.ResultsWe describe a method for sequencing near full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform and test it using DNA from human skin swab samples. Proof of principle of the approach is demonstrated, with the generation of 1,604 sequences greater than 1,300 nt from a single Nano MiSeq run, with accuracy estimated to be 100-fold higher than standard Illumina reads. The reads were chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection.ConclusionsThis method could be scaled up to generate many thousands of sequences per MiSeq run and could be applied to other sequencing platforms. This has great potential for populating databases with high quality, near full-length 16S rRNA gene sequences from under-represented taxa and environments and facilitates analyses of microbial communities at higher resolution.

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