Parallelized sample processing and library prep

Patent metadata

Field	Value
Publication number	US-2022017953-A1
Application number	US-202117333792-A
Country	US
Kind code	A1
Filing date	May 28, 2021
Priority date	Feb 20, 2020
Publication date	Jan 20, 2022
Grant date	—

Abstract

Described herein are methods, kits and systems for sample enrichment, multi-step library preparation, sample normalization, detection of sample biomolecules and combinations thereof. Enrichment and multi-step library preparation is described in the context of microfluidic workflows. Sample barcoding methods and kits are described for increasing sample throughput while reducing background in negative samples. Integrated microfluidic devices comprising sample processing unit cells coupled to an array of reaction sites are provided for integrated workflows.

First claim

1 - 247 . (canceled)

248 . A method of detecting the presence of at least one of a plurality of alleles of a gene in a sample, the method comprising:
a) preamplifying a gene by PCR to obtain a preamplified sample such that a plurality of alleles of the gene would be amplified by the same preamplification primer pair; b) separating the preamplified sample into a plurality of reaction sites; and c) detecting a cycle threshold (CT) of each of the plurality of alleles in a separate reaction site of the plurality of reaction sites, wherein detection is by qPCR and wherein each allele is specifically amplified with a different allele-specific primer pair.

249 - 250 . (canceled)

251 . The method of claim 248 , wherein at least step b) and c) are performed on a microfluidic device, wherein the microfluidic device comprises an array of reaction sites, wherein individual reaction sites of the array of reaction sites comprises an assay chamber and a sample chamber; and wherein sample inlets provide sample to the sample chambers and assay inlets provide assay reagents to the assay chambers

252 . (canceled)

253 . The method of claim 248 , wherein at least one of step a), b) and c) is performed on a microfluidic device wherein the microfluidic device comprises: an array of reaction sites; and a plurality of sample processing unit cells comprising a plurality of sample processing sites, wherein the unit cell is in fluidic communication with a plurality of different reagent inlets; wherein sample inlets to the array are downstream of the plurality of sample processing sites of the plurality of unit cells.

254 . The method of claim 253 , wherein the microfluidic device comprises a column configured to retain beads.

255 . The method of claim 254 , wherein the gene is a viral gene, and further comprising capturing a viral RNA sequence of the gene on the beads through hybridization to a ssDNA sequence prior to step a).

256 . The method of claim 255 , further comprising eluting the viral RNA sequence and preamplifying the eluted viral RNA sequence to produce a cDNA sequence of the gene prior to step a) of preamplifying.

257 . The method of claim 256 , further comprising reverse transcribing the viral RNA sequence captured on the beads to produce a cDNA sequence of the gene prior to step a) of preamplifying.

258 . The method of claim 254 , further comprising capturing viral particles of the sample with antibody conjugated to the beads prior to step a), wherein the gene is a viral gene.

259 . (canceled)

260 . The method of claim 248 , wherein the gene is a viral gene, the method further comprising reverse transcription of viral RNA to produce a cDNA sequence of the gene prior to step a) of preamplifying.

261 . The method of claim 259 , wherein the gene is a viral gene, and the sample is a SARS-CoV- 2 sample.

262 . The method of claim 248 , wherein one allele has a single nucleotide polymorphism compared to another allele and is amplified in step c) with an allele-specific primer pair that is specific to a point mutation.

263 . The method of claim 248 , wherein one allele has an insertion or deletion compared to another allele and is amplified in step c) with an allele-specific primer pair that is specific to the insertion or deletion.

264 . The method of claim 248 , wherein the sample is a biological sample such as a blood sample, saliva, a nasal swab, or derived from solid tissue.

265 . The method of claim 248 , further comprising detecting the presence of at least 4 alleles,

266 . (canceled)

267 . The method of claim 248 , wherein qPCR of the alleles in step c) uses the same probe across the plurality of reaction sites in which different alleles of the gene are detected.

268 . (canceled)

269 . The method of claim 267 , wherein the probe specifically hybridizes to a sequence of the gene conserved across the different alleles.

270 . The method of claim 248 , wherein step a) of preamplifying further comprises amplifying additional genes with additional preamplification primer pairs.

271 . The method of claim 270 , wherein the additional primer pairs include a primer pair specific for the SARS-CoV-2 N1 gene or the SARS-CoV-2 N2 gene.

272 . The method of claim 248 , wherein the alleles include at least two SARS-CoV-2 spike S1 alleles.

273 . The method of claim 248 , wherein at least one allele is amplified in step c) with two allele-specific primers specific for different variant sites of the allele.

274 . The method of claim 248 , wherein at least two alleles are amplified in step c) with the same reverse primer or the same forward primer.

275 . The method of claim 248 , further comprising step d) of identifying the presence of an allele in the sample based on a difference in the CT values (dCT) of the allele and another allele of the plurality of alleles.

276 . The method of claim 275 , wherein the plurality of alleles comprises at least one wildtype allele and two or more mutant alleles.

277 . The method of claim 276 , wherein the presence of each of the mutant alleles is identified in step d) based on whether a dCT between the wildtype allele and the mutant allele is above or below a predetermined dCT threshold.

278 . The method of claim 275 , wherein the predetermined dCT threshold is greater than 3 or less than −3.

279 . (canceled)

280 . The method of claim 275 , wherein the presence of a mutant allele is identified in step d) and the mutant allele is present at 10% or less of a wildtype allele in the sample.

281 . The method of claim 275 , further comprising identifying the presence of a virus based on detection of one or more additional genes preamplified in step a) and detected by qPCR in step c).

282 . The method of claim 281 , further comprising identifying a viral load based on the CT of one or more additional genes preamplified in step a) and detected by qPCR in step c).

283 . The method of claim 280 , wherein the one or more additional genes comprise at least one of the SARS-CoV-2 N1 gene and the SARS-CoV-2 N2 gene.

284 . The method of claim 275 , wherein the CT value of at least one of the alleles of the plurality of alleles is used to report a viral load, wherein the gene is a viral RNA gene.

285 - 306 . (canceled)

Assignees

• Fluidigm Corp

Inventors

• Phelan Michael L
• Kubu Christopher J
• Fowler Brian
• Sun Gang
• Patel Nikita
• Ramalingam Naveen

Classifications

• C12Q1/6851 Primary

  Quantitative amplification · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/70

  involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)} · CPC title