US 9,809,851 B2
Systems and methods for sequencing in emulsion based microfluidics
John Healy, Acton, MA (US)
Assigned to Bio-Rad Laboratories, Inc., Hercules, CA (US)
Filed by GnuBIO, Inc., Cambridge, MA (US)
Filed on May 29, 2014, as Appl. No. 14/290,867.
Claims priority of provisional application 61/828,582, filed on May 29, 2013.
Prior Publication US 2015/0024945 A1, Jan. 22, 2015
Int. Cl. G06F 19/20 (2011.01); C12Q 1/68 (2006.01); C12N 15/10 (2006.01); G06F 19/22 (2011.01); G06F 19/18 (2011.01)
CPC C12Q 1/6874 (2013.01) [C12N 15/1075 (2013.01); G06F 19/22 (2013.01); G06F 19/18 (2013.01)] 9 Claims
 
1. A method of identifying primer/target nucleic acid partitions as being generated from a same target nucleic acid partition mixture drop, the method comprising:
providing a plurality of mixture drops, each mixture drop including copies of at least one target nucleic acid;
generating a plurality of reaction partitions from each mixture drop, the reaction partition including one or more primers, wherein one or more marker reagent is present in each reaction partition such that each primer set in a reaction partition is represented by a pre-determined and known unique signal based on the one or more marker reagent identity and/or concentration; and
detecting signal from the droplets with a detector, wherein the signal includes a signal whether at least one primer in a partition hybridizes to a target nucleic acid;
receiving in a computer a data signal obtained by the detector over a time period, the data signal including signals from a plurality of reaction partitions generated from a plurality of mixture drops, each mixture drop corresponding to a cohort of partitions and including copies of at least one target nucleic acid, each partition including one or more primers, wherein the data signal includes data about whether at least one primer in a partition hybridizes to a target nucleic acid;
identifying a hybridization status of each partition based on the respective signal of the respective partition, the hybridization status of a partition indicating whether a primer in the partition hybridized to a target nucleic acid, wherein a signal of a particular partition corresponds to a particular time;
for each of a plurality of particular times in the time period:
for a time window around the particular time:
calculating with the computer an amount of partitions that have contradictory hybridization statuses and include a same primer, thereby obtaining a temporal function;
identifying extrema in the temporal function; and
determining a cohort of successive partitions occurring between corresponding extrema in the temporal function as corresponding to a same mixture drop.