Computational Systems Biology Methods and Protocols.7z

alignment, which affects the accuracy of quantitative analysis. The

sequencing depth determines the number of detected genes, but it

cannot yield additional information over a threshold. One previous

study showed that the threshold of sequencing depth could be one

million reads [4], but this was not supported by a recent study. In

that study, Lin Liu et al. analyzed a colon cancer scRNA-seq dataset

(SRA: SRP113436), and the results showed that the one-million-

read depth was not enough to acquire all the possible transcripts.

The one-million-read depth had been determined as the threshold

due to not considering all kinds of noncoding RNAs (ncRNAs).

For example, all the previous studies did not consider two novel

long ncRNAs (lncRNAs) discovered in human mitochondrial DNA

[5]. Longer (e.g., 125 or 150 bp) and PE reads improve the

alignment quality and the accuracy of quantitative analysis. How-

ever, most of the existing scRNA-seq datasets contain shorter (e.g.,

50 bp) or SE reads. In addition, the existing scRNA-seq datasets are

Table 1
Overview of scRNA-seq protocols [4]

Method CEL-seq2/C1 Drop-seq

MARS-

seq SCRB-seq Smart-seq/C1

Smart-

seq2

Single-cell

isolation

Automated in

the C1

system

Droplets FACS FACS Automated in

the C1

system

FACS

ERCC

Spike-ins

Yes No Yes Yes Yes Yes

UMI 6 bp 8 bp 8 bp 10 bp No No

Full-length

coverage

No No No No Yes Yes

First strand

synthesis

oligo-dT oligo-dT oligo-dT oligo-dT oligo-dT oligo-dT

Second strand

synthesis

RNAseH/

DNA Pol

Template

switching

RNAseH/

DNA Pol

Template

switching

Template

switching

Template

switching

Amplification IVT PCR IVT PCR PCR PCR

Imaging of cells

possible

Yes NoNoNoYes No

Protocol usable

for bulk

Yes No Yes Yes Yes Yes

Sequencing Paired-end Paired-end Paired-

end

Paired-end Single-end Single-end

Library cost/

cell

~9.5€ ~0.1€ ~1.3€ ~2€ ~25€ ~3/30a

aIn-house produced Tn5/commercial Tn5

Data Analysis in Single-Cell Transcriptome Sequencing 313