Important note for all re-orderers (21.12.2019):
- Eurofins recently optimised the 2-step PCR workflow. Consequently we defined new sample preparation and ordering guidelines.
- This includes the shipment container and the required use of NGS barcodes and the adaptor sequences to be added to the target specific sequences.
- Please read the sample preparation guide carefully.
- In case you have any questions regarding those modifications or need assistance due to the changed ordering process / order sheet, please don't hesistate to contact our support team.
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? Click here
|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.
Table 1. Available libraries and starting material for INVIEW CRISPR Check 2nd PCR*
- 80 - 90 k read pairs per amplicon
- 2 x 300 bp read mode
The CRISPR BioIT 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
- 15 - 20 working days for up to 96 samples.
- 3 additional working days for the bioinformatics analysis
* For less starting material please contact us.
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.