Systems and methods for processing optically tagged beads
The present disclosure provides methods and systems for generating and processing optically tagged beads. A nucleic acid probe comprising a fluorescent tag may be hybridized to a bead to generate an optically tagged bead. The nucleic acid probe hybridized to the optically tagged bead may be detectable by one or more signals and may be substantially unreactive to one or more chemical reactions performed with, or in the presence of, the optically tagged bead.
1 . A method, comprising:
(a) hybridizing a bead and a probe to generate an optically-tagged bead, wherein the optically-tagged bead comprises a binding sequence,
wherein the probe hybridizes to the binding sequence,
wherein the probe comprises: (i) a fluorescent tag; and (ii) at a 3′ end of the probe, a moiety selected from the group consisting of a fluorophore, a carbon spacer, a biotin molecule, an inverted terminal base, a sequence that destabilizes hybridization between the bead and the probe, and an unlocked nucleic acid, and
wherein the moiety renders the probe hybridized to the bead at least 60% less reactive in a base incorporation reaction as compared to an equivalent nucleic acid probe lacking the moiety; and
(b) detecting one or more signals indicative of the fluorescent tag from the optically-tagged bead,
wherein the optically-tagged bead comprises: i) a molecule comprising the binding sequence hybridized to the probe, and ii) a nucleic acid barcode molecule, wherein the molecule is different from the nucleic acid barcode molecule.
2 . The method of claim 1 , wherein the probe comprises the fluorescent tag at a 5′ end of the probe.
3 . The method of claim 1 , further comprising, prior to (a), subjecting a nucleic acid probe comprising the fluorescent tag to conditions sufficient to modify the nucleic acid probe to yield the probe.
4 . The method of claim 3 , further comprising adding the moiety to the nucleic acid probe.
5 . The method of claim 1 , further comprising conducting the base incorporation reaction with or in a presence of the optically-tagged bead.
6 . The method of claim 5 , wherein the base incorporation reaction is an extension reaction.
7 . The method of claim 5 , wherein (b) is performed prior to conducting the base incorporation reaction with or in the presence of the optically-tagged bead.
8 . The method of claim 5 , wherein (b) is performed subsequent to conducting the base incorporation reaction with or in the presence of the optically-tagged bead.
9 . The method of claim 1 , wherein the optically-tagged bead comprises a gel bead.
10 . The method of claim 1 , the method further comprises determining a parameter of the optically-tagged bead based on the one or more signals.
11 . The method of claim 10 , wherein the optically-tagged bead comprises a plurality of primers and wherein the parameter is a concentration of the plurality of primers on the optically-tagged bead, and wherein the probe comprises a probe sequence configured to hybridize to a sequence common to the plurality of primers.
12 . The method of claim 10 , wherein the parameter is a size of the optically-tagged bead.
13 . The method of claim 1 , further comprising, subsequent to (a), partitioning the optically-tagged bead into a partition among a plurality of partitions.
14 . The method of claim 13 , further comprising using the one or more signals to assess a bead occupancy of the partition.
15 . The method of claim 13 , wherein the partition is a droplet or a well.
16 . The method of claim 1 , further comprising, subsequent to (a), co-partitioning the optically-tagged bead with an analyte in a partition among a plurality of partitions.
17 . The method of claim 1 , wherein the carbon spacer comprises a 3 carbon atom spacer, a 6 carbon spacer, a 9 carbon spacer, or an 18 carbon spacer.