This protocol is suitable for tagged amplicons from 600 bp to 15 kb.

Library preparation through the PacBio protocol and PacBio sequencing.

Amplicon generation

This initial amplification of the gene target is performed at your laboratory and it consists of the generation of long amplicons using one of the following approach

Long amplicon with barcode

The PCR amplification targeting the region of interest is conducted using primers that include unique barcodes. This method generates amplicons through a single PCR step, significantly reducing associated biases. Additionally, each amplicon contains a unique sequence, allowing for pooling of multiple amplicons. Please contact our team if you need further information.

Please download the scheme here

Long range PCR with M13 approach:

This protocol is a cheaper approach that allows to perform, pool and sequence up to 384 amplicons against your genomic region of interest:

We perform the first PCR step to amplify the genomic region (or regions) of interest and to add the specific M13-tag in the amplicons.
The second PCR step is necessary to add a combination of barcodes that uniquely tagged each amplicon.

Please download the scheme here

Library Preparation

Regardless the type of approach chosen to generate the amplicons, the last step is the library construction using PacBio SMRTbell Preparation Kit.

Standard protocol: To view samples specifications, sequencing recommendation and samples shipping guidelines, please download the corresponding SP-Doc.

SP-Doc / Long Amplicon sequencing

Fasteris | Defined tool to sequence amplicons of all sizes