NGSelect Amplicon

Please find below a table that should help you to decide for the best sequencing option:

Just let us do the barcoding and pooling work for you

NGSelect Amplicon 2nd PCR starts from PCR samples prepared in your lab with universal adaptors (TruSeq adaptors). Eurofins Genomics performs a 2nd PCR step, the index PCR. This PCR step introduces indexed sequencing adaptors nescessary for sample discrimination for pooled sequencing.

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 2nd PCR:

• No need for your own barcoding - only one primer pair per target region
• Cost-effective outsourcing of the remaining steps
• Easy primer design and primer ordering through Eurofins

Starting material

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)

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)

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

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 340 - 760 bp. For full length sequencing please plan with a max. amplicon length of 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 100-270 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 100 - 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.