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makeIndexFqs.py

A Python script for extracting index sequences from FASTQ read headers and writing them to separate index FASTQ files. Supports two different FASTQ format modes: 3lvl (ScaleRNA v1) and QS (QuantumScale).

Overview

This script processes FASTQ files and extracts index sequences from the read headers to create separate index FASTQ files (I1 and I2). It's designed to work with ScaleBio sequencing data and supports different barcode formats used in various kit versions.

Usage

python makeIndexFqs.py <read1_fastq> [options]

Basic Command

python makeIndexFqs.py sample_R1_001.fastq.gz --outDir ./fastqDir --mode 3lvl

Command Line Options

Option Description Default Required
--outDir Output directory . No
--mode FASTQ format mode: 3lvl or QS 3lvl No
--no-index2 Don't extract Index2 read False No

Modes

3lvl Mode (ScaleRNA v1)

Format: Standard Illumina FASTQ headers with index sequences in the attributes field.

Header Example:

@VH02171:15:2227VLHNX:1:1101:19144:1000 1:N:0:GCTCTCGCCT+TCGGATTCGG

Index Extraction:

  • Index1 (I1 or i7): Extracted from the 4th colon-separated field before the +
  • Index2 (I2 or i5): Extracted from the 4th colon-separated field after the +

Usage:

python makeIndexFqs.py sample_R1_001.fastq.gz --mode 3lvl --outDir ./fastqDir

QS Mode (QuantumScale)

Format: QuantumScale format with partial index1 in read name and full indices in attributes.

Header Example:

@LH00659:241:22T7WLLT4:1:1101:42065:1140:CTGTCCTAATGGGGTTACCGAAGA 1:N:0:TNCAGACA+GTTCGATA

Index Extraction:

  • Index1 (I1 or i7): Concatenation of attribute index1 + read name index (cell barcode)
  • Index2 (I2 or i5): Extracted from attributes after the + (PCR barcode)

Usage:

python makeIndexFqs.py sample_R1_001.fastq.gz --mode QS --outDir ./fastqDir

Output Files

The script generates compressed FASTQ files:

  • I1 file: Contains Index1 sequences (e.g., sample_I1_001.fastq.gz)
  • I2 file: Contains Index2 sequences (e.g., sample_I2_001.fastq.gz) - only if --no-index2 is not used

Examples

ScaleRNA v1 Processing

# Process ScaleRNA v1 data
python makeIndexFqs.py experiment_R1_001.fastq.gz --mode 3lvl --outDir ./fastqDir

QuantumScale Processing

# Process QuantumScale data
python makeIndexFqs.py quantum_R1_001.fastq.gz --mode QS --outDir ./fastqDir

Notes

  • The script automatically handles gzip compression/decompression
  • Quality scores are set to a default value (ASCII 37, offset 33)
  • Index sequences are validated for length consistency across all reads
  • It's important to keep the I1/I2 files together in the same directory as R1/R2 fastqs