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FASTQ Quality Control Tutorial

What is FASTQ Quality Control?

FASTQ files contain raw sequencing data from next-generation sequencing (NGS) instruments. Quality control analysis helps you understand if your sequencing run was successful and if the data is suitable for downstream analysis.

Step-by-Step Guide

Navigate to the FASTQ Tool

Go to genesyslifebio.com/fastq/ or click the "FASTQ Quality Control" card from the homepage.

Upload Your FASTQ File

Click the upload area or drag and drop your FASTQ file. Supported formats include .fastq and .fq files up to 50MB.

If you don't have a FASTQ file, click "View sample results" to see an example analysis.

Configure Analysis Options

  • Number of reads to display: Choose how many example reads to show (default: 10)
  • Reference sequence: Optional - provide a reference for alignment visualization

Run the Analysis

Click "Analyze FASTQ File" to start the analysis. Processing typically takes 5-30 seconds depending on file size.

Review Your Results

The results page shows:

  • Overall quality score with interpretation
  • Read length distribution chart
  • Base composition and GC content
  • Quality score distribution
  • Individual read details with quality visualization

Understanding Quality Scores

Phred quality scores indicate the probability of a base being called incorrectly:

  • Q30+ (Excellent): Less than 1 in 1000 chance of error
  • Q20-29 (Good): Less than 1 in 100 chance of error
  • Q10-19 (Fair): May require quality filtering
  • Q0-9 (Poor): High error rate, consider resequencing

CRISPR Indel Analysis Tutorial

What is CRISPR Indel Analysis?

CRISPR gene editing can introduce insertions or deletions (indels) at target sites. This tool analyzes sequencing data from edited samples to quantify editing efficiency and characterize the types of modifications.

Step-by-Step Guide

Navigate to the CRISPR Tool

Go to genesyslifebio.com/crispr/ or click the "CRISPR Indel Analysis" card from the homepage.

Upload Your Data

Upload a FASTQ file containing sequences from your CRISPR-edited sample.

Enter Reference Sequence

Paste the wild-type (unedited) sequence of your target region. This should include:

  • The PAM site
  • At least 20bp on each side of the expected cut site
  • The entire region you amplified for sequencing
Example: ATCGATCGATCGATCGATCGNGGCGATCGATCGATCGATCG

Select PAM Sequence

Choose the appropriate PAM for your CRISPR system:

  • NGG: SpCas9 (most common)
  • NNGRRT: SaCas9
  • NNNNGATT: NmeCas9
  • TTN: Cas12a/Cpf1

Analyze Results

The analysis will show:

  • Overall editing efficiency (% of reads with indels)
  • Distribution of insertion vs deletion events
  • Most common indel patterns
  • Visual alignment of edited sequences
  • PAM site location and cut site prediction
For accurate results, ensure your reference sequence exactly matches the amplified region and that your sequencing covers the entire potential edit window.

Interpreting Your Results

FASTQ Quality Metrics

Quality Score Distribution

A good sequencing run typically shows:

  • Most reads with average quality >Q30
  • Consistent quality across read length
  • Few reads in the "Poor" category

GC Content

Expected GC content varies by organism:

  • Human: ~40-45%
  • E. coli: ~50%
  • Extreme values (<30% or >70%): May indicate contamination or technical issues

CRISPR Editing Metrics

Editing Efficiency

Typical editing efficiencies:

  • Cell lines: 50-95%
  • Primary cells: 20-80%
  • In vivo: 5-50%

Indel Patterns

Common observations:

  • Small deletions (1-10bp) are most common
  • Insertions are typically 1bp duplications
  • Large deletions may indicate off-target cutting

Frequently Asked Questions

What file formats are supported?
We support standard FASTQ files (.fastq, .fq) up to 50MB. Files can be uncompressed or gzip-compressed (.fastq.gz).
Is my data secure?
Yes. Files are processed in temporary memory and immediately deleted after analysis. We don't store any uploaded data or results.
Can I analyze paired-end reads?
Currently, our tools analyze single-end reads or individual files from paired-end sequencing. For paired-end analysis, upload R1 and R2 files separately.
What sequencing platforms are supported?
Our tools work with FASTQ files from any sequencing platform including Illumina, Oxford Nanopore, PacBio, and Ion Torrent.
How do I export my results?
Use your browser's print function (Ctrl/Cmd + P) to save results as PDF, or copy individual charts and data tables.
Can I use these tools for clinical diagnostics?
Our tools are for research use only and should not be used for clinical decision-making without proper validation.

Need More Help?

If you have questions not covered here, please contact our support team at support@genesyslifebio.com