Direct Colony Sequencing

Direct Colony Sequencing services utilize rolling circle amplification (RCA) to enable Sanger sequencing of bacterial clone or phage sample templates without the need for plasmid preparation. RCA generates DNA for sequencing by random hexamer priming of circular templates. GENEWIZ RCA protocols can handle sequencing projects of any size and are optimized to produce high quality reads from both bacterial colonies and glycerol stocks as well as phage plaques and supernatants. Direct Colony Sequencing is also extremely effective when low amounts of DNA starting material are available (e.g. low copy plasmids).

What is colony sequencing?

Colony sequencing is a method in which plasmid DNA (pDNA) is extracted and amplified directly from microbial colonies, allowing for sequencing data to be produced without the need for plasmid purification. In addition to saving time by eliminating plasmid preparation steps, another key benefit of colony sequencing is its flexibility with the starting sample. Any size plasmid, with a high or low copy plasmid template, can be sequenced with direct colony sequencing. Direct colony sequencing is commonly used for screening complimentary DNA (cDNA) libraries and site-directed mutagenesis.

RCA Sequencing Data in 24 Hours**

Using RCA for DNA amplification expedites the Sanger sequencing process and allows your data to be delivered within 24 hours** of sample receipt at Azenta.

Rolling Circle Amplification Sequencing

RCA was identified in natural systems as a DNA replication mechanism frequently employed in plasmid and viral propagation. When random hexamers are coupled with a strand displacing DNA polymerase, many concatenated template copies are generated with no need for thermocycling (Dean et al., 2001). The synthesized strands are displaced after one round of RCA and are free for additional rounds of primer binding. Exponential amplification is achievable if the RCA reaction contains primers complementary to both template strands

HIGHER YIELD

Scale Up Your Plasmid DNA Preparation

Now offering up to 5 g yields for high-copy, stable plasmids

RCA Sequencing

After lysis, bacterial samples are combined with dNTPs, polymerase, and random hexamers. The hexamers anneal to the circular template, and replication initiates at many sites simultaneously.

Polymerase is utilized due to its high stability, processivity, and high fidelity. This allows prolonged strand displacement over multiple rounds of amplification.

As the newly synthesized strands are displaced, a cascade of priming events facilitates exponential amplification of the template.

Upon completion the sample is ready for Sanger sequencing.

Dean, Frank B. et al. “Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification.” Genome Research 11.6 (2001): 1095–1099.

Direct Colony Sequencing Features & Benefits

Time and cost savings: no need to grow culture and prepare DNA

High-quality data: >600 bases of Phred20 reads

Project design and consultation: Ph.D.-level customer support throughout the project

Fast turnaround: receive your Sanger data within 24 hours** of sample receipt at Azenta

Online ordering: 24/7 online ordering and data retrieval system

GENEWIZ universal primers: select from a wide range of primers available at no additional cost

**24-hour turnaround time guaranteed for maximum of 96 samples; excludes phage samples

RCA Sequencing Applications

shRNA cloning and library screening; Eliminate plasmid preps and sequence colonies directly.

DNA methylation analysis: Save time & obtain high-quality reads by cloning PCR products directly and sequencing the bacterial colonies.

Antibody phage display library validation: Direct phage sequencing for faster screening and validation.

Ensure Complete Plasmid Validation on Your First Attempt

with Oxford Nanopore Sequencing

SAMPLE SUBMISSION OPTIONS

Sample Submission Options

Glycerol Stock*	Send 1.5 ml tubes or 96-well plates; samples must be shipped on dry ice. Please label with media type.
Bacterial Colonies*	Array colonies in a grid on the agar plate and circle the colonies requiring sequencing. Numerically label each colony. Note that unnumbered plates will be processed as random pick. If using liquid culture, submit samples in 96-well plate with strip caps. Wrap agar plates in parafilm, and provide a cushion to protect against potential damage.
Phage Supernatant*	Please submit 50 ul of phage supernatant in 8-strip PCR tubes or 1.5 ml microcentrifuge tubes clearly labeled with sample name.
Phage Plaque	Circle and number the plaques you submit for sequencing. For transport, wrap agar plates in parafilm and provide cushion to protect against potential damage.

*Please note: RCA protocol is intended for circular templates <10 kb; linear templates will not be amplified sufficiently for direct Sanger sequencing.
Longer circular templates, or circular templates carried in bacterial strains with functional endA may not be reliably amplified, contributing to variable results

Please visit our Sample Submission Guidelines for bacteria and phage templates for additional details.

TECHNICAL RESOURCES

Sanger Sequencing Quick Tips Guide

Learn from the Sanger experts how you can get the most of your DNA Sequencing. Whether you're troubleshooting a tough sequencing reaction, perfecting PCR visualization, or learning to clean up and refine your PCR products, these resources are designed to help you get the best results.
• Volume 1: Producing Robust, Single-band PCR Product
• Volume 2: PCR Clean-up of Single-band PCR
• Volume 3: Salvaging Nonspecific PCR Products
• Volume 4: PCR Visualization by Gel Electrophoresis
• Volume 5: Troubleshooting a Bad Sequencing Reaction from a PCR Template

Blog | Troubleshooting DNA Templates with Sanger Sequencing

Sanger sequencing is highly effective for testing small targeted genomic regions and for validating results from NGS. However, as with all sequencing technologies, there are limitations and needs for troubleshooting. Read on to learn about the three basic troubleshooting steps we recommend to start with.

Webinar | From Base Pairs to Breakthroughs: Understanding Sanger and Oxford Nanopore Sequencing

While Sanger sequencing remains the gold standard for accurate DNA sequencing, advancements in sequencing technology, such as Oxford Nanopore Technology (ONT), have provided a cost-effective, long-read alternative for analysis. In this workshop, delve into the considerations of each technique and discover how to interpret their resulting sequencing data effectively so you can capture the benefits of both approaches for your specific needs.

Blog | Analyzing Sanger Sequencing Data

The output for Sanger sequencing is typically a chromatogram, also known as a trace or ab1 file, and a text-based sequence file. Although the latter may seem to hold all the relevant information—after all, the point of sequencing is to get a sequence—the former can't be ignored. Here, we provide a guide to understanding Sanger sequencing data.

HOW TO ORDER

*Samples must arrive at the GENEWIZ New Jersey laboratory before 10:00 am EST to qualify for Same Day service. Note that direct-sequencing templates are not available for our Same Day service.