In WeiSong-bio/roryk-bcbioSinglecell: Single-Cell RNA-Seq Utilities

inDrop sample preparation

FASTQ structure information

• v1: Original design
  • _R1: metadata read
  • _R2: biological read
• v2: Inversion of v1
• v3: Summer 2016 redesign, requiring manual demultiplexing
  • _R1: biological read
  • _R2: first half of the gel barcode
  • _R3: library index
  • _R4: second half of the gel barcode, the UMI, and a fraction of the polyA tail

This study used the [inDrop][] v3 design.

FASTQ demultiplexing

We demultiplexed the raw BCL run files into FASTQ format using [bcl2fastq][]. This step generates files with the following suffixes:

• _R1: 61 bp read 1, transcript
• _R2: 8 bp index read 1 (i7), single cell barcode
• _R3: 8 bp index read 2 (i5), library index
• _R4: 14 bp read 2, barcode and unique molecular identifiers (UMIs)

We ran this on [Orchestra][] with the following settings:

{bash bcl2fastq, echo=TRUE, eval=FALSE} module load seq/bcl2fastq/2.17.1.14 bcl2fastq --use-bases-mask y*,y*,y*,y* \ --mask-short-adapter-reads 0 \ --minimum-trimmed-read-length 0

WeiSong-bio/roryk-bcbioSinglecell documentation built on July 6, 2019, 12:03 a.m.