Methods and systems for processing polynucleotides

A method for nucleic acid sequencing, comprising:

• (a) providing a plurality of partitions, wherein a partition of the plurality of partitions comprises:
• (i) a plurality of ribonucleic acid (RNA) molecules from a single cell;
• and
• (ii) a particle comprising nucleic acid barcode molecules coupled thereto,
• wherein the nucleic acid barcode molecules coupled to the particle comprise a first subset and a second subset,
• wherein the first subset comprises a partition-specific barcode sequence and a first primer region comprising a poly-T sequence, and
• wherein the second subset comprises the partition-specific barcode sequence and a second primer region comprising a sequence that is complementary to a sequence encoding a T cell receptor (TCR) or B cell receptor (BCR) constant region;
• (b) using the plurality of RNA molecules and the first subset of nucleic acid barcode molecules to generate a first plurality of barcoded nucleic acid molecules, each comprising:
• 1) the partition-specific barcode sequence or a complement thereof, and 2) a sequence of a messenger RNA (mRNA) molecule of the plurality of RNA molecules or a complement thereof;
• and using the plurality of RNA molecules and the second subset of nucleic acid barcode molecules to generate a second plurality of barcoded nucleic acid molecules, each comprising:
• 1) the partition-specific barcode sequence or a complement thereof, and 2) a TCR or BCR variable region-encoding sequence or a complement thereof;
• wherein the generating the first plurality of barcoded nucleic acid molecules and the second plurality of barcoded nucleic acid molecules comprises:
• (i) hybridizing the first subset of nucleic acid barcode molecules to poly-A sequences in mRNA molecules of the plurality of RNA molecules in the partition, and hybridizing the second subset of nucleic acid barcode molecules to sequences encoding TCR or BCR constant regions in mRNA molecules of the plurality of RNA molecules in the partition;
• (ii) extending the first and second subsets of the nucleic acid barcode molecules using the hybridized mRNA molecules as template with a reverse transcriptase having terminal transferase activity to generate first and second barcoded cDNA molecules comprising 3′ terminal sequences added by the reverse transcriptase;
• (iii) hybridizing the 3′ terminal sequences of the first and second barcoded cDNA molecules to hybridization regions of template switching oligonucleotides (TSOs), and
• (iv) extending the first and second barcoded cDNA molecules using the TSOs as templates, thereby generating the first plurality of barcoded nucleic acid molecules and the second plurality of barcoded nucleic acid molecules;
• (c) amplifying barcoded nucleic acid molecules of the first plurality of barcoded nucleic acid molecules or derivatives thereof to generate an unenriched sequencing library;
• and
• (d) amplifying barcoded nucleic acid molecules of the second plurality of barcoded nucleic acid molecules or derivatives thereof to generate an immune receptor-enriched sequencing library.