bcbioSinglecell: Import and analyzebcbio single-cell RNA-seq data

Michael J. Steinbaugh, PhDmsteinbaugh@hsph.harvard.edu

July 26, 2017

http://bioinformatics.sph.harvard.edu/bcbioSinglecell

Cell population-level analysis facilitates cell typeand differentiation cascade profiling

Stegle et al., Nat Rev Genet 16:133-145 (2015)

How to get here?Barcode filteringQuality control

§ Native HPC support for single-cell RNA-seq analysis.§ Uses the umis package internally to process barcodes.§ Quantifies counts using either rapmap or kallisto. § Outputs transcript-level counts in MartrixMarket format.§ bcbioSinglecell: R package interface to scRNA-seq data.

• Automates sample import into R as a SummarizedExperiment.• Saves counts as sparse matrix, using Matrix package.• Provides a number of quality control functions and RMarkdown

templates for clustering workflows (e.g. Seurat, monocle).• Data integration between Python and R!

https://bcbio-nextgen.readthedocs.io/http://bioinformatics.sph.harvard.edu/bcbioSinglecell/

Barcode samples

Sequence

FASTQ

Quasi-map reads

Process barcodes

inDropDrop-seqSureCell

Illumina

bcl2fastq

umis

rapmap, kallisto

scaterQuality control

Clustering

Differential expression

Trajectorymonocle

SCDE

Seurat

10X ChromiumCell Ranger

Load into R

bcbio

bcbioSinglecell

bcbioSCDataSet S4 class

gene-level counts dgCMatrix

Gene annotable

tx-level counts dgCMatrix

slotNames()

assays

metadata

callers

colData

NAMES

elementMetadata

bcbio run settings SimpleList

hdf5?

cellular barcode metrics

Ensembl gene identifiers

sparse = lower memory overhead

SummarizedExperiment

QC metrics and barcode filteringReads per barcode Cell counts

Gene detection Mitochondrial abundance

bcbioSCSubset S4 class

SummarizedExperiment

• min UMIs• min/max genes• max mito ratio• min novelty

seuratSCESet CellDataSet

bcbioSCDataSet

filter()

as()

bcbioSCSubset

scater Seurat monocle

Harvard Chan Bioinformatics Core

Mary Piper

John Hutchinson

Lorena Pantano

Brad Chapman

Peter Kraft

Rory Kirchner

Meeta Mistry Radhika Khetani

Shannan Ho Sui

Victor BarreraMichael Steinbaugh