High-efficiency, high-quality genotyping results of CRISPR-edited cells
Leveraging our history in automated genome engineering, EditCo has applied our editing expertise and high-throughput optimization with NGS edit genotyping to deliver superior CRISPR edits. We achieve high efficiency and accuracy of genotyping results for CRISPR-edited cells through high-throughput automated NGS library preparation and proprietary data analysis.
Single guide knockouts
For downstream genotyping analysis, raw fastq files are pre-processed to retain high quality reads mapped back to the target region. Across 1,536 replicate experiments, where an average of 96.5% of bases had a Q score ≥ 30, genotyping result accuracy of a heterozygous single guide knockout clone fell within 3.7% of the expected 50% (horizontal dashed line) -6 bp deletion (pink dots) observed and 50% -3 bp deletion (purple dots) observed at 1000 mapped NGS reads (vertical dashed line).
Multi-guide knockouts
Multi-guide knockout edits are inherently difficult to evaluate by traditional sequencing methods due to large fragment deletions and often requiring multiple attempts in order to successfully perform genotyping analysis. NGS (purple bar) had a 100% first pass success rate (y axis) at genotyping a multi-guide knockout CRISPR edit pool (52/52 attempts) compared to Sanger sequencing (pink bar) at 52% (27/52), ensuring quality genotyping results will be delivered faster.
Sensitivity of NGS genotyping results
Knock-in edited pools are a mixed population of edited and unedited cells, thus downstream clonal isolation is reliant on accurate determination of the proportion of edited cells with the desired knock-in event. A +14-bp small knock-in edited pool was titrated against wild type and assessed by Sanger sequencing and NGS for levels of knock-in (y axis) compared to predicted levels (blue bars). At every titration level (x axis), NGS (purple bars) is more sensitive than Sanger sequencing (pink bars).
Genotype correlation between Sanger (ICE) and NGS across different edit types
Knockout and Knock-in scores are in high agreement (62% fall within a KO/KI score of +/- 5) between Sanger sequencing and ICE analysis and NGS and EditCo’s proprietary internal analysis software for edited cells, including single guide clones and pools (single guide KO, blue bars), Tags and Small knock-ins <100 bps (small KI, pink bars), and multi-guide pools (multi-guide KO, purple bars). Consistent with the Small KI titration data, overall NGS is more sensitive, reflected in a shift toward higher knockout and knock-in scores (49.7% of knockout/knock-in scores are + values along x axis) over Sanger sequencing genotyping results (9% of knockout/knock-in scores are - values along x axis).
Glossary of key terms:
- MiSeq – an Illumina NGS sequencing platform.
- %Q30 – The percentage of bases with a quality score of 30 (Q30) or higher.
- Q30 – A quality score of 30 indicates the base call is correct 999/1,000 of the time.
- Read depth – the amount of unique DNA strands sequenced onboard the NGS platform.
- Combinatorial Dual Index (CDI) – an indexing strategy which leverages the use of index primers in combination to determine sample identity. A particular index primer may be used more than once, but the combination of index primers are always unique to each sample being sequenced.
Frequently Asked Questions:
General information
What's the difference between Sanger sequencing and Next Generation Sequencing (NGS)?
- Sanger sequencing is an older, though robust sequencing technology in the right applications. Sequencing with Sanger involves the collection of nucleotide frequencies for a given sample. For complex, heterogenous mixtures, this technology does not always give the resolution needed.
- Conversely, NGS sequencing is a much newer sequencing technique which reads thousands of single DNA strands in parallel. This strategy allows for improved data confidence and much better resolution, especially when applied to complex mixtures of diverse CRISPR edits.
What's EditCo's process for NGS preparation and analysis?
- EditCo’s NGS preparation process is performed on integrated automation instrumentation with an array of QC feedback loops to ensure sample quality and process robustness from end to end. Libraries are sequenced with Combinatorial Dual Index (CDI) Nextera primers, normalized to ensure consistently adequate read depth, and are run on Illumina’s MiSeq platform internally.
- As part of EditCo's software pipeline, raw data is pre-processed by quality filtering, adapter trimming, merging paired-end, and mapping reads. Processed reads are then analyzed by EditCo's proprietary software to genotype editing outcomes.
Can EditCo's ICE tool be used to read the sequence file meant for NGS?
- ICE is only able to analyze Sanger sequencing files; NGS data analysis is not compatible with ICE analysis.
Order Related Information
What information is included in the QC report?
- All the genotyping information corresponding to your order will be available in a single .pdf QC document. This document will include all the relevant information to your order as well as the NGS sequencing information, index size distribution and sequence alignment.
How can I read the NGS results in my QC report?
- The Engineered Cells QC Report User Guide document includes information on how to interpret the information included in your QC document, including NGS key words and definitions.
Does EditCo use the reference genome or the wild type sequence?
- We sequence the wild type of every sample and align against this wild type sample.
Will this affect the overall turn-around-time of my order?
- The turn-around time for your order should not be affected with this process improvement.
- EditCo’s NGS process was built with a variety of QC checks, and process parameters were designed to ensure sequencing from end-to-end completes, even for difficult gene target regions, before your cells are shipped.
Will EditCo provide Sanger primers if I want to confirm my edit using Sanger sequencing?
- Yes, Sanger primers will be provided upon request.
Will EditCo provide NGS primer sequences if I am working with GKO/Libraries and I want to confirm my genotype using NGS?
- Yes, NGS primers will be provided upon request.
Can I still use ICE using Sanger sequencing to validate or analyze my CRISPR edits?
- Yes, ICE is still available to analyze your edits using Sanger sequencing.
Recommendations for Genotyping
- For SpCas9 single-guide knockouts and knock-in CRISPR edits, if you want to obtain genotypes using Next Generation Sequencing (NGS), we recommend using CRISPResso.
- Alternatively, you can analyze SpCas9 single-guide, multi-guide, and knock-in CRISPR edits using EditCo’s ICE tool, which relies on Sanger sequencing. Notably, EditCo’s ICE tool is currently the only publicly available option for analyzing multi-guide derived CRISPR edits.
- Our Genotyping protocol includes all the necessary information to genotype your cells.
