BAC clone characterization
The CNRGV aims to offer a comprehensive service from plant DNA extraction to the characterization of genetic information underpinning a trait of interest. To meet this goal the CNRGV currently utilizes Next Generation Sequencing (NGS) technologies to effectively analyze BAC clones of interest. We focus in particular on the quality of the assembly and on finding various solutions that enable us to achieve this.
PacBio sequencing (Pacific Biosciences : up to 60kb reads to reach the perfect assembly)
This NGS method available since few years produces 9kb average read length and up to 1Gb of data for 1 SMRT cell, the longest reads can be up to 60kb. Depending on the characteristics of the BAC clones to sequence (complexity of the region, BAC clones overlapping,…), we use different sequencing strategies. BAC clones are pooled with or without tagging, sequenced together on one SMRT cell with the P6C4 chemistry.
The sequencing is carried out in collaboration with the Sequencing Platform of Toulouse which has a PacBio RSII sequencer. The DNA of each BAC clone is prepared in CNRGV using the best protocol to obtain high quality samples. The CNRGV is also responsible for the preparation of sequencing libraries.
A new pipeline has been specifically developed by the CNRGV to analyse PacBio data and reach the best quality assembly. Each BAC is then individualized by matching their BES (BAC Ends Sequences) on the ends of assembled contigs.