towards isoform resolution single-cell transcriptomics for

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Towards Isoform Resolution Single-Cell Transcriptomics for Clinical Applications Using Highly Accurate Long-Read SequencingAbstract #: 1873Elizabeth Tseng1, Jason G. Underwood1, Arjun Scott Nanda2, Vijay Ramani2, Scott N. Furlan3

1PacBio, 1305 O’Brien Drive, Menlo Park, CA 94025 2UCSF, San Francisco, CA 3Fred Hutchinson Cancer Research Center, Seattle, WA

Improving scIso-Seq Throughput on PacBio Systems PacBio Sequencing & Deconcatenation Single-Cell Deconvoluation With Short or Long Reads

• PacBio Iso-Seq method generates full-length transcript sequences up to ~15kb with high accuracy (>99.9%)

• 10X single-cell systems produce ~50% TSO-TSO artifact cDNA• Using TSO artifact depletion and cDNA concatenation, we

achieve ~6X throughput, or 8-9 million full-length cDNA molecules per SMRT Cell 8M for the 10X single-cell platform

• We applied to this throughput-improvement method to 10X single-cell libraries sequenced on PacBio Sequel II systems

• Demonstrated cell BC concordance with matching short read libraries

• Full-length isoform information revealed distinct expression levels in T cells not observable through 3’ tagging methods

scIso-Seq Throughput Improvement Methodology

Sample A Sample B

HiFi Reads 2,557,092 3,174,724

Reads with cDNA primers 2,151,948 2,726,226

Deconcatenated cDNAs 7,853,190 8,519,673

Hypothetical cDNAs w/out TSO depletion and concatenation

1,075,974 1,363,113

Effective Throughput Increase ~7.2X ~6.2X

Distribution of Concatemers per Long ReadSample A

Transcript Classification using SQANTI3

Cell BC concordance, PacBio vs. Illumina

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B MemoryB NaiveBasophilsCD14 MonoCD16 MonoCD4 MemoryCD4 NaiveCD8 EffectorCD8 MemoryCD8 NaiveCD8 TRB−V9

cDCISG15_High TregMAITMultipletsNeutrophilNKpDCProliferatingRBCTreg

Long Reads

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Short Reads

15388 short reads/cell936 cDNAs/cell

Knee plot, all BC PacBio

Short reads calls8386 single cells

(10X Cell Ranger)

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Short Reads

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MultipleNonePB.30915.37PB.30915.4PB.30915.5PB.30915.7

iso30915ISG20 Isoforms Assigned to Single Cells

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iso30915

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iso30915

T celllineages

T cells express4 common isoforms

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Short Reads

Sample A (5’ library) read schema

Assigning Isoforms to Single CellsISG20 : Interferon Stimulated Exonuclease Gene 20

The Complete Diversity of ISG20 Isoforms Expressed in CD4 Naïve Cells

CD8 Naïve Cells Prefer from the Downstream TSS

GENCODE Reference

towards isoform resolution single-cell transcriptomics for

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