Parallelized sample processing and library prep
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.
1. A method of detecting a 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 an array plurality of reaction sites on a microfluidic device; and
c) detecting a cycle threshold (CT) of each of the plurality of alleles in a separate reaction site of the array of reaction sites, wherein detection is by qPCR, wherein at least two alleles are amplified with a same forward primer, wherein detection is by qPCR wherein each allele is specifically amplified with a different allele-specific reverse primer, wherein qPCR of the alleles in step c) uses a same probe across the array of reaction sites in which different alleles of the gene are detected,
wherein the microfluidic device comprises: the array of reaction sites; and a plurality of sample processing unit cells, each sample processing unit cell comprising a plurality of sample processing sites,
wherein each of the plurality of sample processing sites is upstream from and in fluidic communication with a respective sample inlet of a reaction site of the array of reaction sites,
wherein each of the plurality of sample processing sites is downstream from a cleanup column, wherein the cleanup column is downstream from a unit cell sample inlet,
wherein each sample processing unit cell further comprises a valve and a pump configured to move flow
i) downstream from the unit cell sample inlet, through the cleanup column, and to the plurality of sample processing sites, and
ii) upstream from the plurality of sample processing sites, through the cleanup column, to the unit cell sample inlet, and
wherein step a) is optionally performed on the microfluidic device.
2. The method of claim 1 , wherein individual reaction sites of the array of reaction sites comprise an assay chamber that is in fluid communication with a respective assay inlet to receive assay reagents and a sample chamber that is in fluid communication with the respective sample inlet of the reaction site.
3. The method of claim 1 , wherein the cleanup column is configured to retain beads.
4. The method of claim 3 , 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).
5. The method of claim 4 , 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.
6. The method of claim 5 , 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.
7. The method of claim 3 , 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.
8. The method of claim 1 , 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.
9. The method of claim 1 , wherein the gene is a viral gene, and the sample is a SARS-COV-2 sample.
10. The method of claim 1 , 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.
11. The method of claim 1 , 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.
12. The method of claim 1 , wherein the sample is a biological sample, wherein the biological sample is a blood sample, a saliva, a nasal swab, or derived from a solid tissue.
13. The method of claim 1 , further comprising detecting the presence of at least 4 alleles.
14. The method of claim 1 , wherein the probe specifically hybridizes to a sequence of the gene conserved across the different alleles.
15. The method of any claim 1 , wherein step a) of preamplifying further comprises amplifying additional genes with additional preamplification primer pairs.
16. The method of claim 15 , wherein the additional preamplification primer pairs include a primer pair specific for a SARS-COV-2 N1 gene or a SARS-COV-2 N2 gene.
17. The method of claim 1 , wherein the alleles include at least two SARS-COV-2 spike S1 alleles.
18. The method of claim 1 , wherein at least one allele is amplified in step c) with two allele-specific primers specific for different variant sites of the allele.
19. The method of claim 1 , 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.
20. The method of claim 19 , wherein the plurality of alleles comprises at least one wildtype allele and two or more mutant alleles.
21. The method of claim 20 , 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.
22. The method of claim 21 , wherein the predetermined dCT threshold is greater than 3 or less than-3.
23. The method of claim 19 , 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.
24. The method of claim 19 , 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).
25. The method of claim 24 , 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).
26. The method of claim 23 , wherein the one or more additional genes comprise at least one of a SARS-COV-2 N1 gene and a SARS-COV-2 N2 gene.
27. The method of claim 19 , 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.