Hybrid targeted and whole transcriptome amplification
Disclosed herein include systems, methods, compositions, and kits for the labeling of nucleic acids targets. In some embodiments, the methods comprise the use of target-specific primers and target-non-specific primers during library preparation (e.g., hybrid library preparation). In some embodiments, the methods increase the abundance of select low abundance targets during cDNA library preparation and/or sequencing library preparation.
1. A method of labeling nucleic acid targets, the method comprising:
(a) hybridizing a plurality of nucleic acid targets with a plurality of oligonucleotides each comprising a first universal sequence and a barcode sequence, wherein the plurality of nucleic acid targets comprises two or more nucleic acid targets;
(b) extending the plurality of oligonucleotides hybridized to the plurality of nucleic acid targets to generate a plurality of first strand barcoded polynucleotides;
(c) synthesizing a plurality of second strand barcoded polynucleotides using the plurality of first strand barcoded polynucleotides as templates to generate a plurality of double-stranded barcoded polynucleotides;
(d) adding the sequence of an adaptor to the plurality of double-stranded barcoded polynucleotides, wherein the adaptor comprises a second universal sequence;
(e) amplifying the plurality of double-stranded barcoded polynucleotides using:
(e1) primers capable of hybridizing to the first universal sequence and the second universal sequence, thereby generating a first plurality of barcoded amplicons comprising sequences of the nucleic acid targets, the first universal sequence and the second universal sequence, and
(e2) primers capable of hybridizing to two or more of the plurality of nucleic acid targets and a primer capable of amplifying the first universal sequence, thereby generating a second plurality of barcoded amplicons comprising sequences of the two or more of the plurality of nucleic acid targets and the first universal sequence; and
(f) performing random priming and extension using random primers and the first plurality of barcoded amplicons and the second plurality of barcoded amplicons as templates.
2. The method of claim 1 , wherein (f) performing random priming and extension comprises adding sequences of binding sites of sequencing primers and/or sequencing adaptors, complementary sequences thereof, and/or portions thereof, to the first plurality of barcoded amplicons and the second plurality of barcoded amplicons.
3. The method of claim 1 , comprising (g) obtaining sequencing reads of the first plurality of the barcoded amplicons and/or the second plurality of the barcoded amplicons.
4. The method of claim 1 , wherein the first plurality of barcoded amplicons corresponds to at least 10%, at least 50%, or at least 90%, of the mRNAs of a single cell.
5. The method of claim 1 , wherein the second plurality of barcoded amplicons correspond to at most 5%, at most 10%, or at most 20%, of the mRNAs of a single cell.
6. The method of claim 1 , wherein the adaptor is a single-stranded polynucleotide.
7. The method of claim 1 , wherein the adaptor is a double-stranded polynucleotide.
8. The method of claim 1 , wherein each one of the plurality of amplicons comprises at least part of the first universal sequence, at least part of the second universal sequence, or both.
9. The method of claim 1 , wherein (d) adding the sequence of the adaptor to the plurality of double-stranded barcoded polynucleotides comprises ligating the adaptor to the plurality of double-stranded barcoded polynucleotides.
10. The method of claim 1 , wherein (d) adding the sequence of the adaptor to the plurality of double-stranded barcoded polynucleotides comprises adding the sequence of the adaptor to the plurality of double-stranded barcoded polynucleotides by nucleic acid extension or amplification.
11. The method of claim 1 , wherein the two or more of the plurality of nucleic acid targets comprises mRNAs of a low-expressing gene, wherein the percentage of nucleic acid targets that represent mRNAs of the low-expressing gene is at most 5%.
12. The method of claim 1 , wherein the total number of occurrences of the two or more of the plurality of nucleic acid targets comprises mRNAs of at most 1%, at most 5%, or at most 10%, of the mRNAs of a single cell.
13. The method of claim 1 , wherein the sequence of the adaptor is added to 5′ end, 3′ end, or both of the plurality of double-stranded barcoded polynucleotides.