RNA sequencing methods
Described herein are methods for determining a sequence of a region of interest from an mRNA molecule. Sequenced polynucleotides can include a barcode region, a homopolymer region (e.g., a poly-A region), and a target region associated with the mRNA molecule. According to some methods, the barcode region omits the same base present in the homopolymer region. According to some methods, extension of the primer used for sequencing is stalled within the homopolymer region. According to some methods, sequencing flow cycles and the different barcode regions of the polynucleotides configured are such that the primer is extended to the end of the barcode region across the plurality of polynucleotides before being extended into the homopolymer region. According to some methods, two primers or a cleavable primer is used to separately sequence the barcode region and the target region.
1 . A method of determining a sequence of a region of interest from an mRNA molecule, comprising:
(a) hybridizing a plurality of polynucleotides derived from mRNA with a primer to form a plurality of hybridized templates, the polynucleotides comprising a barcode region, a homopolymer region comprising a plurality of contiguous and identical bases, and a target region comprising a sequence associated with a region of interest from an mRNA molecule;
(b) determining the sequence of the barcode region using labeled nucleotides and unlabeled nucleotides at a first proportion of labeled nucleotides to total nucleotides;
(c) extending the primer using labeled nucleotides complementary to the base present in the homopolymer region at a second proportion of labeled nucleotides to total nucleotides, wherein the second proportion is greater than the first proportion, wherein primer extension stalls within the homopolymer region;
(d) extending the primer to the end of the homopolymer region using unlabeled nucleotides complementary to the base present in the homopolymer region; and
(e) determining the sequence of the target region using labeled nucleotides.
2 . The method of claim 1 , wherein the labeled nucleotides and unlabeled nucleotides used in step (b) comprise non-terminating labeled nucleotides and non-terminating unlabeled nucleotides.
3 . The method of claim 1 , wherein the labeled nucleotides used in step (c) comprise non-terminating labeled nucleotides.
4 . The method of claim 1 , wherein the unlabeled nucleotides used in step (d) comprise non-terminating unlabeled nucleotides.
5 . The method of claim 1 , wherein the labeled nucleotides used in step (e) comprise non-terminating labeled nucleotides.
6 . The method of claim 1 , wherein the sequence of the target region is determined using labeled nucleotides and unlabeled nucleotides.
7 . The method of claim 1 , wherein 50% or fewer of the nucleotides used in step (b) or (e) are labeled.
8 . The method of claim 1 , wherein greater than 50% of the nucleotides used in step (c) are labeled.
9 . The method of claim 1 , wherein 0.1% or fewer of the nucleotides used in step (d) are labeled.
10 . The method of claim 1 , wherein all of the nucleotides used in step (d) are unlabeled.
11 . The method of claim 1 , wherein step (d) comprises repeatedly removing unincorporated nucleotides and adding fresh nucleotides one or more times until the primer is extended to the end of the homopolymer region.
12 . The method of claim 1 , wherein the target region of each polynucleotide is associated with a unique barcode region.
13 . The method of claim 1 , wherein different bases of the labeled nucleotides used to determine the target region are discretely used.
14 . The method of claim 1 , wherein different bases of the labeled nucleotides used to determine the barcode region are discretely used.
15 . The method of claim 1 , wherein the bases in the homopolymer region of the polynucleotides are adenine or thymine bases.
16 . The method of claim 1 , wherein the homopolymer region of the polynucleotides comprises as least 8 contiguous and identical bases.
17 . The method of claim 1 , wherein the homopolymer region of the polynucleotides comprises as least 50 contiguous and identical bases.
18 . The method of claim 1 , wherein the polynucleotides are cDNA molecules.
19 . The method of claim 1 , wherein the region of interest of the mRNA molecule comprises a coding region of the mRNA molecule.
20 . The method of claim 1 , wherein the region of interest of the mRNA molecule comprises a 3′-untranslated region or a 5′-untranslated region of the mRNA molecule.