Complete sequencing of BAC clone inserts is now routine.

Many researchers need perfectly accurate sequence of just a few BAC clones, from a few regions of interest.

Quick Strategic Overview with PacBio:

  1. Make High Quality NGS grade BAC DNA (minimizing host E. coli genomic DNA) – or you may provide us with ~10µg of ultra pure BAC DNA per clone.
  2. Make a high quality PacBio library (with 20Kb fragments and tags to allow combining several BAC’s in each PacBio run).
  3. Run the PAcBio reads through the HGAP DNA assembly pipeline to obtain as few contigs as possible.
  4. Genomic Content will impact assembly results. Our experience of typical results for 10 random BAC clones:  7 BAC’s will have a single contig (no gaps), 2 BAC’s will have 1 or 2 gaps and one BAC will have more than 3 gaps.

SEQUENCE THE MOST CHALLENGING BACS

Scientists often use bacterial artificial chromosome (BAC) sequencing to close gaps in existing genome assemblies, or as a method of targeted sequencing to fine-map regions of interest. BAC clones containing GC- or AT-rich regions or repeat structures can be challenging for short-read sequencing technologies.

Complete BACs with confidence

With the long reads and uniform coverage of S

ingle Molecule, Real-Time (SMRT) Sequencing, we can sequence and assemble finished BACs with high consensus accuracy – often into a single contig per BAC.

SMRT Sequencing on PacBio Systems gives you the ability to:

  • Reconstruct medically and developmentally important structures within complex regions, such as the Y chromosome
  • Finely map genes of interest with structural variation and haplotype information for evolutionary and agricultural studies
  • Generate platinum-level genome assemblies
  • Resolve historically difficult-to-sequence regions in draft genomes
  • Pool dozens of BACs in a single SMRT Cell for efficient and cost-effective sequencing

Workflow: Using BACs to close coverage of the most complex regions

Library Preparation

  • NO PCR is used with Pac Bio SMRTbell libraries
  • Prepare long-insert SMRTbell libraries (20Kb fragments)
  • Enrich for longest inserts with size selection options
 

SMRT Sequencing on PacBio Systems

  • Through our partners, you can take advantage of the Sequel System to reduce project costs
  • Attain consensus accuracies >99.999% by avoiding mapping and systematic errors
  • Scale throughput based on project needs; recommend 50-fold coverage for high-quality de novo genome assemblies
  Data Analysis with SMRT Analysis

  • Generate de novo assemblies with HGAP within SMRT Analysis