Please find below a table that should help you to decide for the best sequencing option:
Comparison |
ONT Lite Clonal Amplicons |
Amplicons on ONT |
Amplicons on NovaSeq (5M) |
Amplicons on MiSeq (60k) |
MiSeq full run (2x300 bp) |
MiSeq full run (2x250 bp) |
Sequencing technology |
GridION |
GridION |
NovaSeq |
MiSeq |
MiSeq |
MiSeq |
Amplicon size
|
1 kb – 25 kb |
> 270 bp |
150 – 270 bp |
300 – 570 bp |
300 – 570 bp |
280 bp – 470 bp |
Delivery time
|
3 – 5 days |
From 20 days |
From 20 days |
From 12 days |
From 15 days |
From 15 days |
Primer demand |
Without inline barcodes: One primer pair per target region |
With inline barcodes*: e.g. designed by NGS Adapter Ligation Oligos
Layout: see below
Without inline barcodes: One primer pair per target region.
|
One primer pair per target region, incl adapter sequence (as defined in the sample submission guide)
Primer Design Tool available at: NGSgrade Oligos for MiSeq
|
Advantages |
Short TAT, Great price (€ 15) |
Long & complex amplicon sequences, Phasing in long amplicons possible |
Ideal for short amplicon regions to be sequenced |
Best price, especially for small projects |
Attractive for larger projects, e.g with sample pool |
Disadvantages |
Only for clonal amplicons |
Homopolymer regions & methylation sites hard to sequence
Q22 for most bases
|
Small amplicon size |
You cannot send a sample pool, but single samples |
Not cost efficient for small projects |
* de-multiplexing from inline barcodes to be done by customer
# phasing = variants originated from same amplicon can be identified
Layout for inline barcodes (using adaptor ligation on NovaSeq or ONT)
- One forward primer for each sample and target region
- One rev primer per target region (e.g. 20 samples and 1 target region needs 21 primers)
Just let us do the barcoding and pooling work for you
NGSelect Amplicon on MISeq starts from PCR samples prepared in your lab with universal adaptors (TruSeq adaptors). We then perform indexing of your samples and subsequent sequencing on Illumina MiSeq.
For sample preparation you just need to add a sequence adaptor to your target specific primers that is given in the sample submission guide. For amplification of the same target region in a series of samples, you thus need only one primer pair per target.
Highlights NGSelect Amplicons on MiSeq:
- No need for your own barcoding - only one primer pair per target region
- Cost-effective outsourcing of the remaining steps
- Amplicon size: 280 - 570 bp
- You can choose between 60k read pairs data output, as well as full MiSeq runs (2 x 300 bp or 2 x 250 bp)
- Easy primer design and primer ordering through Eurofins
Starting material
Library Type
|
Starting Material
|
Illumina MiSeq |
25 µl of purified amplicon, preferred in EB buffer (PCR product)*
Amplicon size (not including the universal adaptors) between 280 - 570 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 1.5 ml safe-lock tubes.
|
Sequencing possibilities
Standard:
- Guaranteed 60 k read pairs per amplicon
- 2 x 300 bp read mode
Custom:
- Full MiSeq flow cell (2 x 300 bp or 2 x 250 bp)
Adaptor ligation on Illumina
The adaptor ligation approach can be applied for short amplicons for Illumina sequencing.
With Illumina amplicon sequencing, extremely high coverage suitable for discovery of somatic mutations in complex DNA samples can be achieved. Proprietary protocols and years of NGS expertise help provide high quality data with extraordinary fast delivery times.
Highlights of NGSelect Amplicon
- Large selection of Illumina sequencing options
- Highly sensitive NGS workflows with reduced bias
- Flexible data packages, starting from 5M reads
- Multiplexing of up to 192 Illumina samples
- For multiplexed Illumina sequencing: Eurofins Primer Design to save your time and ensure best practice multiplexing
Starting Material
Library Type
|
Starting Material
|
Illumina NovaSeq |
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 for sequencing with 5M reads pairs 2 x 150 bp
|
Sequencing packages
- Guaranteed 5M read pairs (10 million reads) per package, 2 x 150 bp
Bioinformatics analysis
- Merging of overlapping paired-end reads
- Data sorting according to tags designed by Eurofins Genomics
- Data sorting according to customer supplied tag
Sequencing of large and/or repetitive amplicons.
Product description:
- Library preparation:
- ONT adapter ligation (multiplexing depends on amplicon size and preferred coverage, max. 96 samples per Flow Cell)
- Sequencing:
- Technology: Oxford Nanopore
- Output read length: Equal to fragment length (can be up to several kbp)
- Throughput is dependent upon the quality of sample submitted
- Approx. 7 Gb per Flow Cells (dependent on DNA quality and length)
- Deliverables:
- Raw Nanopore Amplicon data (FastQ)
- BioIT on request (e.g. Variant analysis VCF-file)
- Input required (dependent on multiplexing):
- OD 260/280: 1.8oOD 260/230: 2.0-2.2
- Max. amplicon size: 20 kb
- For 1 sample:
- Optimal: >25ng/μl in 60 μl
- For 2-48 samples:
- Size range of different amplicons should be similar (+/-10%)
- Optimal: >25ng/μl in 30 μl
Additional bioinformatics option:
ONT Amplicon Bioinformatics Analysis
- Sequence Quality Assessment
- Amplicon consensus generation
- Amplicon variant sites
- Reporting & Consultancy
Deliverables
- Raw sequencing reads (fastq.gz)
- Amplicon consensus sequence (fasta)
- Analysis report (HTML)
It's as simple as can be
- Define the loci(s) you want to amplify and design target specific primer sequences that amplify a region of 280 - 570 bp.
- Upload your target specific sequence on our corresponding oligo order page and let Eurofins do the automatic and validated primer design.
- Oder NGSgrade primers and prepare amplicons with the designed primers.
- Check amplification success on a gel and send purified amplicons to Eurofins.
Any questions or support required? Please contact us.
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 150-270 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 150 - 270 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.