COMPOSITIONS AND METHODS FOR SAMPLE PROCESSING
First Claim
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1. A method of preparing a barcode library, comprising:
- providing a plurality of separate first bead populations;
attaching a first oligonucleotide comprising a first barcode sequence segment to the separate first bead populations, wherein each separate first bead population comprises a different first barcode sequence segment attached thereto;
pooling the separate bead populations to provide a first pooled bead population;
separating the first pooled bead population into a plurality of second bead populations;
attaching a second oligonucleotide comprising a second barcode sequence segment to the first oligonucleotide already attached to the second bead populations, wherein each of the plurality of second bead populations comprises a different second barcode sequence segment; and
pooling the plurality of second bead populations to provide a second pooled bead population comprising a barcode library.
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Abstract
This disclosure provides methods and compositions for sample processing, particularly for sequencing applications. Included within this disclosure are bead compositions, such as diverse libraries of beads attached to large numbers of oligonucleotides containing barcodes. Often, the beads provides herein are degradable. For example, they may contain disulfide bonds that are susceptible to reducing agents. The methods provided herein include methods of making libraries of barcoded beads as well as methods of combining the beads with a sample, such as by using a microfluidic device.
209 Citations
43 Claims
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1. A method of preparing a barcode library, comprising:
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providing a plurality of separate first bead populations; attaching a first oligonucleotide comprising a first barcode sequence segment to the separate first bead populations, wherein each separate first bead population comprises a different first barcode sequence segment attached thereto; pooling the separate bead populations to provide a first pooled bead population; separating the first pooled bead population into a plurality of second bead populations; attaching a second oligonucleotide comprising a second barcode sequence segment to the first oligonucleotide already attached to the second bead populations, wherein each of the plurality of second bead populations comprises a different second barcode sequence segment; and pooling the plurality of second bead populations to provide a second pooled bead population comprising a barcode library. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
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34. A method of preparing a barcode library, comprising:
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providing a first pooled bead population comprising a plurality of different first bead populations, each different first bead population having a different first oligonucleotide attached thereto each different first oligonucleotide comprising a different first barcode sequence segment; separating the first pooled bead population into a plurality of second bead populations; attaching a second oligonucleotide comprising a second barcode sequence segment to the first oligonucleotide already attached to the second bead populations, wherein each second bead population comprises a different second barcode sequence segment; and pooling the second bead populations to provide a second pooled bead population comprising a barcode library. - View Dependent Claims (35, 36, 37, 38, 39, 40)
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41. A barcode library, comprising a plurality of different oligonucleotides, each different oligonucleotide comprising:
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a first barcode sequence segment selected from a first set of barcode sequence segments; a second barcode sequence segment selected from a second set of barcode sequence segments; and a linking sequence joining the first barcode sequence segment and the second barcode sequence segment, the linking sequence having from about 2 to about 6 nucleotides in length and being selected from a set of linking sequences. - View Dependent Claims (42, 43)
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Specification