Tailed amplicon sequencing

Tailed-amplicon sequencing project example

Three samples (pools) containing 5 amplicons each (left) or a more sophisticated design where 15 amplicons are pooled into one sample (right). If the library contains, for example, 94 samples, 470 (left) or 1410 (right) amplicons can be sequenced in this way. The data is demultiplexed into individual samples (94 files) upon delivery. Further differentiation takes place according to the length of the product, the sequence of primers or other identifiers used. Image courtesy of Mr. Luis Perera Fernández.

Overview

Should you need a really low-cost sequencing strategy for hundreds or even thousands of DNA fragments at a time, this Tailed Amplicon approach is the right choice for you. Although its name mentions amplicons, it is suitable for dsDNA fragments bearing specific tails at both 5´ and 3´ends, obtained not necessarily by PCR exclusively. In fact, it is compatible with DNA fragments generated by tagmentase cleavage (Illumina Tagment DNA TDE1 Enzyme) or DNA fragments having specific tails ligated. In any case, DNA fragments supplied must bear specific tails as outlined below! Why? Because in our sequencing lab, these specific tails are utilized in order to add another set of tails (adapters) needed for sequencing. At this step, unique index combination chosen from our large stock is introduced and allows high level of multiplexing. This approach enables you to focus on any region of your interest. Also it leaves the initial sample preparation under your full control enabling you to work with reagents of your choice but without the need of purchasing large number of indexed adaptors. Instead, we supply these and prepare the sequencing library for subsequent sequencing using any Illumina platform we offer.

Procedure

Step 1 in YOUR lab - You perform PCR to amplify regions of interest and pool all amplicons from the same sample into a single pool. For example, should you have 94 samples and 5 different amplicons per sample, you perform 5 x 94 PCR reactions and pool amplicons per sample. Samples must be further purified and diluted to a uniform concentration according to the valid Sample submission guidelines.

Step 2 in SEQme NGS lab - We perform 2nd PCR step to add sequencing indices and adapters, perform all necessary QC steps and pool all 94 samples into a single library and proceed with sequencing.

How to design primers

Append to 5’ end of your primers: 

• Forward: 5’ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-[locus specific sequence]
• Reverse: 5’ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-[locus specific sequence]

Laboratory processing

You perform 1st PCR step using primers above. Then, in our lab your samples will be processed as follows:

• Amplification of DNA fragments supplied by using tailed-primers (2nd PCR step)
• PCR product quantification and pooling to achieve optimal balanced sequencing yield
• Quantification of libraries by qPCR
• Sequencing on Illumina systems 

Results 

• The reads obtained will be sorted according to the combination of indexes into files representing individual samples and analysis of sequencing quality indicators such as number and length of sequences, phred score, %GC, duplication level, etc. will be performed.
• As output you will receive data in FASTQ format divided into files according to individual samples. Upon request, the outputs can be processed in another form. Please specify this when ordering.

Processing time

Approx. 4 weeks (data analysis not included)

Sample requirements

Follow our Sample submission guidelines.

• Samples must be purified after first PCR and delivered exactly in required concentration! Too low a concentration could lead to a low PCR yield, high concentration to different yields or even inhibition of the reaction (high DNA concentration may be associated with a high concentration of inhibitors). Please provide samples in 0,2 mL strips or 96-well plates.
• It is required that samples are provided with tails (overhangs) as described above, purified from artifacts such as excess primers or adaptors, enzymes etc. A picture of agarose gel showing (representative) DNA fragments as submitted is also needed.
• Total lenght of DNA fragments must be between 200 and 900 bp!

How to order the service

The analysis can be ordered and commissioned online as a package for 24, 48, 94 or 190 samples. If you want to order an analysis of a different number of samples (eg only a few samples or, conversely, thousands), contact us to prepare an individual calculation. 

 ORDER