NGSelect Amplicon Adaptor Ligation
Highest flexibility in amplicon size and data packages
The adaptor ligation approach can be applied for both, short amplicons for Illumina sequencing and amplicons up to 6 kb for PacBio long read sequencing.
With Illumina amplicon sequencing, extremely high coverage suitable for discovery of somatic mutations in complex DNA samples can be achieved. PacBio amplicon sequencing offers the chance to cover the whole length of the PCR products, eliminating the need for any assembly. Proprietary protocols and years of NGS expertise help provide high quality data with extraordinary fast delivery times.
Highlights of NGSelect Amplicon
- Sequencing of amplicons ranging from 100 bp to 6 kb in size
- Large selection of Illumina sequencing options or PacBio sequencing options
- Highly sensitive NGS workflows with reduced bias
- Flexible data packages, starting from 5M reads
- Multiplexing of up to 192 Illumina samples or 10 PacBio samples possible
- For multiplexed Illumina sequencing: Eurofins Primer Design to save your time and ensure best practice multiplexing
You need oligos to prepare your Amplicons? Click here
|Illumina adapter ligation library
100 - 200 ng PCR product / amplicons (up to 100 µl, concentration > 1 ng/µl
Amplicon size range should ideally range from 150 bp to 270 bp (absolute min. 80 bp, absolute max. 650 bp) for sequencing with 5M reads pairs 2 x 150 bp
Amplicon size range should ideally range from 270 bp to 570 bp (absolute min. 250 bp, absolute max. 650 bp) for sequencing with 12M reads pairs 2 x 250 bp or 16M read pairs 2 x 300 bp
|PacBio adapter ligation library
Amplicon size range of:
570 bp - 3 kb = 2 µg (up to 40 µl, concentration > 50 ng/µl)
3 kb - 6 kb = 4 µg (up to 40µl, concentration > 100 ng/µl)
- 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
- PacBio data packages
- “Reads of Insert” ~15,000 reads (for long amplicon sizes of 570 bp to 3 kb)
- “Reads of Insert” ~ 3,000 reads (for long amplicon sizes of 3 kb to 6 kb)
- Merging of overlapping paired-end reads
- Data sorting according to tags designed by Eurofins Genomics
- Data sorting according to customer supplied tag
- “Reads of insert” analysis
- Generation of consensus sequences from read inserts
- Demultiplexing and sorting of reads (if applicable)
Starting from less than two weeks, varies depending on individual project aims
*For less starting material, please contact us.
How to prepare your Amplicons for NGSelect Adaptor Ligation (Illumina)
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 regions of 100-570 bp.
- 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 regions of 100 - 570 bp.
- 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 and quantify each amplicon for equimolar pooling
- The use of tagged primers enable multiplexed sequencing of several pooled amplicons within a single data package.