Highest flexibility in amplicon size and data packages
INVIEW CRISPR Check Adaptor Ligation starts from short PCR samples prepared in your lab that you optionally provide with molecular tags. Eurofins Genomics continues with adaptor ligation on each PCR sample or sample pool and thus introduces sequencing adaptors necessary for Illumina sequencing.
Highlights of INVIEW CRISPR Check Adaptor Ligation
- Sequencing of (Wildtype) amplicons up to 500 bp
- Large selection of Illumina sequencing options
- Flexible data packages, starting from 5M read pairs packages
- Pooling of up to 192 samples
- Bioinformatics pipeline specially designed for identification and quantification of CRISPR events
- Free of charge Eurofins Primer design for application of molecular tags to save your time and to ensure best practice multiplexing
You have a large number of samples that you would like to analyse using this workflow? In these cases we can offer a customised Project.
You have applied the HDR repair mechanism? Please contact us for a customised project discussion.
You Need Oligos to prepare your Amplicons? Click here
Product details
Starting Material
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Library Type
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Starting Material
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| Illumina adapter ligation library |
100 - 200 ng PCR product / amplicons (up to 100 µl, concentration > 1 ng/µl
Amplicon length requirements:
Sequencing with 5M read pairs package(s), 2 x 150 bp:
For assessment of deletions of up to 50 bp and insertions of up to 30 bp, the wildtype amplicon size (not including the tags) should be between 150 and 225 bp.
Sequencing with 12M read pairs package(s), 2 x 250 bp:
For e.g. assessment of deletions of up to 50 bp and insertions of up to 30 bp, the wildtype amplicon size (not including the tags) should be between 325 and 400 bp.
Sequencing with 16M read pairs package(s), 2 x 300 bp:
For e.g. assessment of deletions of up to 50 bp and insertions of up to 30 bp, the wildtype amplicon size (not including the tags) should be between 325 and 500 bp.
Further sample requirements can be found in the sample submission guide.
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Specifications
- Illumina data packages
- Guaranteed 5M read pairs (10 million reads) per package, 2 x 150 bp
- Guaranteed 12M read pairs (24 million reads) per package, 2 x 250 bp
- Guaranteed 16M read pairs (32 million reads) per package, 2 x 300 bp
- Data sorting according to tags designed by Eurofins Genomics
- Data sorting according to customer supplied tag
- CRISPR mutation analysis
The CRISPR mutation analysis evaluates mutations introduced by non-homologous end joining (NHEJ) pathway. Our pipeline is designed for analysing events with single breaks.
- Quality clipping and merging of raw reads
- Mapping to target region(s) and quality filtering of mapped reads
- Clustering based identification and quantification of NHEJ events
- Quantification of mutation rates and of frameshift mutations
- For each sample and target region, detailed reporting of:
- Wildtype and alternate allele sequences including quantification
- Observed mutation rate plots, frameshift classification plots
- Raw data, mapping, and coverage statistics
- Comprehensive report with results and methods sections
- Raw FASTQ files
- Quality clipped and assembled FASTQ files
- Mapped reads in BAM format (raw and filtered)
- Observed wildtype and alternate allele sequences including quantification
- Raw data, preprocessing, mapping, and coverage metrics in CSV format
Delivery time starting from 20 working days.
*For less starting material, please contact us.
How to prepare your Amplicons for INVIEW CRISPR Check Adaptor Ligation
It's simpler than ever
For the adaptor ligation protocol, you can either provide plain amplicons for adaptor ligation or make use of our tagging strategy. The latter reduces sequencing costs especially for projects involving more than a few samples.
Option without tagging:
- Define the loci(s) you want to amplify and design target specific primer sequences that amplify the selected region.
- Order target specific NGSgrade oligo primers and prepare amplicons
- Check amplification success on a gel and purify the amplicons
- As no tagging has been performed a minimum of 1 data package has to be ordered per amplicon
Option with tagging:
- Define the loci(s) you want to amplify and design target specific primer sequences that amplify the selected region.
- Upload your target specific sequence(s) on our corresponding oligo order page and let Eurofins do the automatic primer design.
- Order primers and prepare amplicons with the designed primers.
- Check amplification success on a gel & purify individual amplicons or your amplicon pool
- The use of tagged primers enable multiplexed sequencing of several pooled amplicons within a single data package.