Batch Oligo QC Tool for 10,000 Sequences

Validate oligo pools before synthesis by screening up to 10,000 sequences for GC content, Tm spread, secondary structures, homopolymers, length drift, and low complexity regions. Export flagged rows for redesign, then move passing sequences into vendor-specific order formatting. For pool-specific thresholds, open the pre-order oligo pool QC guide or check the Design Guide for best practices.

Before synthesis submission

Paste a FASTA list, one sequence per line, or a vendor-prep CSV. Review GC outside the target range, long homopolymers, Tm outliers, low complexity, length drift, and structure risk. Redesign flagged rows, then use the Vendor Format Adapter for upload files or the Oligo Pool Design Guide when design assumptions need another pass.

Drag and drop a FASTA file here, or click to browse

Max 10,000 sequences, 10MB file size

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Upload a FASTA file or paste sequences to begin quality control analysis.

What Is Batch Sequence Quality Control?

Batch Sequence QC is the systematic analysis of all oligonucleotide sequences in a pool or library before placing a synthesis order. It identifies sequences that are likely to fail during synthesis, have poor functional performance, or introduce bias into downstream experiments. For oligo pools used in CRISPR screens, gene assembly, or mutagenesis libraries, pre-synthesis QC is essential to avoid costly redesign cycles.

Our Batch Sequence QC tool analyzes up to 10,000 sequences simultaneously, checking each for: GC content extremes, homopolymer runs (repeated bases like AAAAA), low-complexity regions, Tm outliers, potential secondary structures, and length violations. Each sequence receives a risk score, and flagged sequences are highlighted with specific failure reasons.

The tool generates a downloadable QC report with summary statistics (pool-wide GC distribution, Tm range, flag frequency) and per-sequence details. This report can be shared with synthesis vendors to discuss design modifications or used internally to guide sequence redesign before ordering.

Use this batch sequence QC page for pool-scale validation before synthesis submission. The QC guides can help set thresholds and interpret vendor or NGS reports; this tool performs the actual sequence screening.

Typical input includes a FASTA list, one sequence per line, or a vendor-prep CSV. Review GC outside target range, long homopolymers, Tm outliers, low complexity, length drift, and structure risk before moving passing rows into vendor-specific order formatting.

How to Use the Batch Sequence QC Tool

  1. Upload your sequences: paste one sequence per line, upload a FASTA file, or import a CSV with sequence names and sequences.
  2. Set QC thresholds: adjust min/max GC content, max homopolymer length, min/max sequence length, and Tm range based on your application requirements.
  3. Click "Run QC" to analyze all sequences. Processing 10,000 sequences takes approximately 2-5 seconds.
  4. Review the summary dashboard: overall pass/fail rate, GC content distribution, Tm distribution, and most common failure reasons.
  5. Examine flagged sequences individually — each flag includes the specific issue and suggested remediation.
  6. Export the QC report as CSV for your records or to share with your synthesis vendor.

Frequently Asked Questions

What QC checks should I perform before ordering an oligo pool?
At minimum, check: (1) GC content distribution — sequences outside 25-75% GC have higher dropout rates; (2) homopolymer runs — runs of 5+ identical bases cause synthesis errors; (3) Tm uniformity — pool Tm CV should be below 3°C for functional assays; (4) length uniformity — most synthesis platforms have strict length limits; (5) secondary structure — stable hairpins (ΔG < -3 kcal/mol) reduce synthesis yield. Our tool checks all of these automatically.
What is an acceptable pass rate for an oligo pool QC?
For most applications, aim for >90% of sequences passing all QC checks. Pools with <80% pass rate should be redesigned. For critical applications like CRISPR screens where library completeness matters, aim for >95% pass rate. Note that even flagged sequences may synthesize successfully — flags indicate elevated risk, not guaranteed failure. However, it is best practice to redesign flagged sequences when possible.
How do homopolymer runs affect synthesis quality?
Runs of 4+ identical bases (e.g., AAAA, GGGG) cause increased deletion rates during phosphoramidite synthesis. Poly-G and poly-C runs are particularly problematic because they form stable secondary structures (G-quadruplexes, i-motifs) that stall the synthesis cycle. For array-based synthesis, homopolymer runs of 6+ bases are a major cause of sequence dropout. We recommend keeping homopolymer runs to 4 bases or fewer.
Can I use this tool to validate CRISPR sgRNA libraries?
Yes, this tool is widely used for CRISPR library validation. For sgRNA libraries, focus on: (1) GC content between 30-70% for each guide; (2) no poly-T runs of 4+ bases (which act as a Pol III terminator and silence the guide); (3) avoid stable secondary structures in the spacer region; (4) verify that all guides are the correct length (typically 20 nt for SpCas9). Set custom thresholds appropriate for your CRISPR system.
What is the difference between Batch Sequence QC and individual sequence analysis?
Individual tools (Tm Calculator, GC Analyzer, etc.) analyze one sequence at a time with deep analysis. Batch Sequence QC processes thousands of sequences simultaneously with a comprehensive but streamlined analysis. Batch QC also provides pool-level statistics (distributions, outliers, uniformity metrics) that are not available from individual tools. Use individual tools for primer design optimization; use Batch QC for library-scale validation before synthesis.

Related Tools

Uniformity Estimator

After QC, estimate your pool's expected representation uniformity and dropout rate.

Error Rate Calculator

Calculate expected synthesis error rates and full-length percentage for your sequences.

GC Content Analyzer

Deep-dive into GC content distribution for individual sequences flagged by Batch QC.

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Related reading

Continue with the page or tool that matches the next decision in your experiment.

Pre-Order Oligo Pool QCOligo Pool Design GuideVendor Format AdapterRead Oligo Pool QC MetricsCRISPR Library Design GuideUser Guide