MOLECULAR DEDUPLICATION ANALYSIS METHODS
The invention provides methods for measuring quantities of mRNA transcripts present in a sample, where sequence information for each molecule is read from what is essentially a random start site within that molecule and in which a short binning index (e.g., about 3 bases) is added to the sequence information. The binning index is useful to resolve any bias arising with the use of the intrinsic sequences to uniquely identify and count the molecules.
1 . A method of measuring gene expression, the method comprising:
sequencing mRNA or cDNA from random start sites of genomic DNA to generate sequence reads each having a unique portion;
attaching a binning index to the sequence reads;
mapping each sequence read to a genomic region;
determining counts of said unique portions per genomic region;
assigning the counts to associated binning indexes;
correcting counts to reduce bias introduced during sample preparation; and
summing corrected counts across the binning indexes for each genomic region to provide an estimated number of the transcripts per genomic region in the sample.
2 . The method of claim 1 , wherein the binning index includes five or fewer bases, preferably three or fewer.
3 . The method of claim 1 , wherein sample preparation comprises:
fragmenting mRNA at the random start sites, annealing oligonucleotides to the fragments, and extending the oligonucleotides to make cDNA.
4 . The method of claim 1 , wherein sample preparation comprises:
annealing oligonucleotides to the mRNA, extending the oligonucleotides to make cDNA copies of the transcripts, and fragmenting the cDNA copies at the random start sites.
5 . The method of claim 1 , wherein the correcting step accounts for a probability of the random start sites being duplicated among the transcripts.
6 . The method of claim 1 , wherein the correction factor accounts for a probability of multiple random start sites per transcript within the sample.
7 . The method of claim 1 , wherein the genomic DNA is prepared by capturing the transcripts with capture oligos linked to beads, wherein each capture oligo includes ′5-linkage to bead, cell barcode, binning index, annealing primer section-3′.
8 . The method of claim 7 , wherein the annealing primer section comprises a poly-T region.
9 . The method of claim 7 , wherein a first portion of the capture oligos linked to the beads include no binning index and a second portion of the capture oligos linked to the beads each include a binning index that each independently consists of 1, 2, or 3 bases.
10 . The method of claim 7 , further comprising isolating a cell with one of the beads in an aqueous partition and lysing the cell to release the transcripts within the partition.
11 . The method of claim 1 , wherein the assigning step comprises writing in memory one or more files that include the counts indexed by the binning indexes and optionally wherein, after the assigning step, for each gene, only the binning indexes, the counts, and the correction factor are used to for the applying and summing step.