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? see below
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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Specification:
- 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
Just let us do the barcoding and pooling work for you
INVIEW CRISPR Check 2nd PCR starts from PCR samples prepared in your lab with universal adaptors. Eurofins Genomics performs a 2nd PCR step, the index PCR. This PCR step introduces indexed sequencing adaptors necessary for sample discrimination for pooled sequencing.
For sample preparation you just need to add a universal sequence adaptor to your target specific primers. For amplification of the same target region in a series of samples, you thus need only one primer pair per target.
Highlights INVIEW CRISPR Check 2nd PCR:
- No need for your own barcoding - only one primer pair per target region
- No need for exact quantification of purification of amplicons
- Cost-effective outsourcing of the remaining steps
- Bioinformatics pipeline specially designed for identification and quantification of CRISPR events
- Easy primer design and primer ordering through Eurofins
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 with full MiSeq runs. Please contact us for a project discussion.
You Need Oligos to prepare your Amplicons? See below
Product details
Starting material
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Library Type
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Starting Material
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| Illumina 2nd PCR library |
25 µl of purified amplicon, preferred in EB buffer (PCR product)*
Amplicon size needs to be adapted to the length of InDels you want to be able to analyse and must allow merging of read pairs. E.g. for assessment of deletions of up to 50 bp and insertions of up to 30 bp, the wildtype amplicon size (not including overhangs 1st PCR) should be between 400 and 500 bp.
Quality check need to be performed by agarose gel electrophoresis (= clear visible band(s) of expected size using 5 µl PCR product).
Samples need to be shipped in 96-well PCR plates with 200 µl well volume.
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Table 1. Available libraries and starting material for INVIEW CRISPR Check 2nd PCR*
Specification:
Illumina data packages:
- Guaranteed 60k read pairs per amplicon
- 2 x 300 bp read mode
Analysis
- 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
Deliverables
- 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
NGSgrade Oligos for Adaptor ligation
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.
NGSgrade Oligos for 2nd PCR
How to prepare your Amplicons for INVIEW CRISPR Check 2nd PCR
It's as simple as it can be
- Define the locus or loci you want to amplify and design target specific primer sequences that amplify the region(s).
- Upload your target specific sequence on our corresponding oligo order page and let Eurofins do the automatic primer design.
- Order NGSgrade primers and prepare amplicons with the designed primers.
- Check amplification success on a gel and send unpurified amplicons to Eurofins.
Do you have more Questions? Please contact us.
Adaptor ligation:
Delivery time starting from 20 working days.
2nd PCR:
- 15 - 20 working days for up to 96 samples.
- 3 additional working days for the bioinformatics analysis