Dataset Information

Construction of Strand-seq libraries in open nanoliter arrays.

ABSTRACT: Single-cell Strand-seq generates directional genomic information to study DNA repair, assemble genomes, and map structural variation onto chromosome-length haplotypes. We report a nanoliter-volume, one-pot (OP) Strand-seq library preparation protocol in which reagents are added cumulatively, DNA purification steps are avoided, and enzymes are inactivated with a thermolabile protease. OP-Strand-seq libraries capture 10%-25% of the genome from a single-cell with reduced costs and increased throughput.

SUBMITTER: Hanlon VCT

PROVIDER: S-EPMC9017222 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Construction of Strand-seq libraries in open nanoliter arrays.

Hanlon Vincent C T VCT Chan Daniel D DD Hamadeh Zeid Z Wang Yanni Y Mattsson Carl-Adam CA Spierings Diana C J DCJ Coope Robin J N RJN Lansdorp Peter M PM

Cell reports methods 20220124 1

Single-cell Strand-seq generates directional genomic information to study DNA repair, assemble genomes, and map structural variation onto chromosome-length haplotypes. We report a nanoliter-volume, one-pot (OP) Strand-seq library preparation protocol in which reagents are added cumulatively, DNA purification steps are avoided, and enzymes are inactivated with a thermolabile protease. OP-Strand-seq libraries capture 10%-25% of the genome from a single-cell with reduced costs and increased through ...[more]

PMID: 35474869

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Project description:The SlipChip platform was tested to perform high-throughput nanoliter multiplex PCR. The advantages of using the SlipChip platform for multiplex PCR include the ability to preload arrays of dry primers, instrument-free sample manipulation, small sample volume, and high-throughput capacity. The SlipChip was designed to preload one primer pair per reaction compartment and to screen up to 384 different primer pairs with less than 30 nanoliters of sample per reaction compartment. Both a 40-well and a 384-well design of the SlipChip were tested for multiplex PCR. In the geometries used here, the sample fluid was spontaneously compartmentalized into discrete volumes even before slipping of the two plates of the SlipChip, but slipping introduced additional capabilities that made devices more robust and versatile. The wells of this SlipChip were designed to overcome potential problems associated with thermal expansion. By using circular wells filled with oil and overlapping them with square wells filled with the aqueous PCR mixture, a droplet of aqueous PCR mixture was always surrounded by the lubricating fluid. In this design, during heating and thermal expansion, only oil was expelled from the compartment and leaking of the aqueous solution was prevented. Both 40-well and 384-well devices were found to be free from cross-contamination, and end point fluorescence detection provided reliable readout. Multiple samples could also be screened on the same SlipChip simultaneously. Multiplex PCR was validated on the 384-well SlipChip with 20 different primer pairs to identify 16 bacterial and fungal species commonly presented in blood infections. The SlipChip correctly identified five different bacterial or fungal species in separate experiments. In addition, the presence of the resistance gene mecA in methicillin resistant Staphylococcus aureus (MRSA) was identified. The SlipChip will be useful for applications involving PCR arrays and lays the foundation for new strategies for diagnostics, point-of-care devices, and immobilization-based arrays.

Dataset Information

Construction of Strand-seq libraries in open nanoliter arrays.

Publications

Construction of Strand-seq libraries in open nanoliter arrays.

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