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FastQ Output

The fastq directory contains FASTQ file generation results, quality control reports, and file assignment information for each sequencing library.

Directory Structure

fastq
├── fastq_file_to_library_assignment.csv
└── fastqc
    ├── QSR-P-SCALEPLEX_1_QSR-P-SCALEPLEX_S553_R1_001_fastqc.html
    ├── QSR-P-SCALEPLEX_1_QSR-P-SCALEPLEX_S553_R1_001_fastqc.zip
    ├── QSR-P-SCALEPLEX_1_QSR-P-SCALEPLEX_S553_R2_001_fastqc.html
    ├── QSR-P-SCALEPLEX_1_QSR-P-SCALEPLEX_S553_R2_001_fastqc.zip
    ├── QSR-P-SCALEPLEX_2_QSR-P-SCALEPLEX_S554_R1_001_fastqc.html
    ├── QSR-P-SCALEPLEX_2_QSR-P-SCALEPLEX_S554_R1_001_fastqc.zip
    ├── QSR-P-SCALEPLEX_2_QSR-P-SCALEPLEX_S554_R2_001_fastqc.html
    ├── QSR-P-SCALEPLEX_2_QSR-P-SCALEPLEX_S554_R2_001_fastqc.zip
    ├── QSR-P-SCALEPLEX_2_QSR-P-SCALEPLEX_S555_R1_001_fastqc.html
    ├── QSR-P-SCALEPLEX_2_QSR-P-SCALEPLEX_S555_R1_001_fastqc.zip
    ├── QSR-P-SCALEPLEX_2_QSR-P-SCALEPLEX_S555_R2_001_fastqc.html
    ├── QSR-P-SCALEPLEX_2_QSR-P-SCALEPLEX_S555_R2_001_fastqc.zip
    └── ... (additional fastqc reports for all samples)

Key Files and Directories

File Assignment (fastq_file_to_library_assignment.csv)

CSV file containing the mapping between FASTQ files and library indices:

  • FASTQ filename: Original sequencing file name
  • Library Index: Corresponding library identifier
  • File path: Location of the FASTQ file

Quality Control Reports (fastqc/)

Quality control reports generated by FastQC for each FASTQ file:

File Naming Convention

<libIndex2>-<sample_name>_<libIndex2>-<sample_name>_S<sample_number>_R<read_number>_001_fastqc.{html|zip}

Example: QSR-P-SCALEPLEX_10A_QSR-P-SCALEPLEX_S553_R1_001_fastqc.html

  • QSR-P-SCALEPLEX: Library index
  • 1 or 2: Bead barcode alias; 1 of 96
  • S553: Sample number (NOT biological sample); arbitrary
  • R1: Read 1 or Read 2
  • 001: Lane number
  • fastqc: Tool identifier

Report Types

Interactive Reports (*_fastqc.html)

  • Interactive quality control reports
  • Visualizations of sequencing quality metrics
  • Detailed analysis of read characteristics
  • Pass/fail warnings for quality thresholds

Compressed Archives (*_fastqc.zip)

  • Compressed versions of HTML reports
  • Include raw data files for custom analysis
  • Can be uploaded to MultiQC for batch analysis

FastQC Quality Metrics

Per Base Sequence Quality

  • Phred Quality Scores: Average quality scores across read positions
  • Quality Distribution: Spread of quality scores at each position
  • Quality Trends: Changes in quality across read length

Per Sequence Quality Scores

  • Overall Quality: Distribution of mean quality per read
  • Quality Thresholds: Pass/fail criteria for quality standards

Per Base Sequence Content

  • GC Content: Guanine-Cytosine content across read positions
  • Base Composition: Distribution of A, T, G, C at each position
  • Bias Detection: Systematic biases in base composition

Per Sequence GC Content

  • GC Distribution: Distribution of GC content per read
  • Expected vs Observed: Comparison with theoretical distributions

Per Base N Content

  • Ambiguous Bases: Frequency of N (unknown) bases
  • Quality Issues: Positions with high N content

Sequence Length Distribution

  • Read Lengths: Distribution of read lengths
  • Length Consistency: Variation in read lengths

Sequence Duplication Levels

  • Duplicate Detection: Frequency of duplicate sequences
  • Library Complexity: Assessment of library diversity

Overrepresented Sequences

  • Adapter Contamination: Detection of adapter sequences
  • PCR Artifacts: Identification of PCR duplicates
  • Contaminant Sequences: Foreign DNA contamination

Adapter Content

  • Adapter Sequences: Detection of sequencing adapters
  • Trimming Recommendations: Suggested adapter trimming

See full documentation here: FastQC

File Organization

Sample Structure

Each sample typically has multiple FASTQ files:

  • R1 files: (Read 1)
  • R2 files: (Read 2)
  • Multiple lanes: If sequencing was performed across multiple lanes

Library Organization

  • Single library: All samples from one library index; these are NOT sample level fastqs
  • Multiple libraries: Different library indices for different experiments
  • Sample multiplexing: Multiple samples per library

Viewing FastQC Reports

Individual FastQC Reports

You can view individual FastQC reports by opening the HTML files directly in a web browser:

# Open individual HTML report in browser
open fastq/fastqc/*_fastqc.html

Batch Analysis with MultiQC

For comprehensive analysis of all FastQC results, see the MultiQC report in the /reports directory:

  • Location: reports/multiqc_report.html
  • Features: Summary dashboard, comparative analysis, interactive plots
  • Benefits: View all samples simultaneously, identify patterns and outliers

See QC Reports for detailed information about MultiQC reports and other quality control outputs.

Need Help?

For more information, please contact support@scale.bio or visit our support website.