Preparation of next-generation sequencing libraries from damaged DNA
Adrian W Briggs1, Patricia Heyn
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. abriggs@genetics.med.harvard.edu
Methods in Molecular Biology (Clifton, N.J.)
|January 13, 2012
Summary
This study introduces an efficient ancient DNA library preparation protocol for next-generation sequencing (NGS). The method enhances DNA conversion and minimizes uracil-induced errors, improving ancient DNA research accuracy.
Area of Science:
- Paleogenomics
- Molecular Biology
- Bioinformatics
Background:
- Ancient DNA (aDNA) research is vital for understanding past life.
- Next-generation sequencing (NGS) combined with target enrichment advances aDNA studies.
- Degraded aDNA, low copy numbers, and uracil lesions hinder sequencing depth and accuracy.
Purpose of the Study:
- To develop a highly efficient protocol for ancient DNA library preparation.
- To integrate uracil lesion removal into the library preparation workflow.
- To streamline the process, reducing hands-on time and purification steps.
Main Methods:
- A novel protocol for converting DNA extracts into adaptor-ligated sequencing libraries.
- Incorporation of an optional uracil removal step during library preparation.
- Utilizes only two spin column purifications, avoiding gel or bead-based methods.
Main Results:
- Achieves highly efficient conversion of DNA fragments into sequencing libraries.
- Effectively removes the majority of uracil miscoding lesions.
- Generates a finished, amplified library in as little as 3-5 hours.
Conclusions:
- The protocol significantly improves ancient DNA library preparation efficiency and accuracy.
- It offers a rapid and streamlined method for generating high-quality sequencing libraries from degraded DNA.
- This advancement facilitates deeper insights from ancient DNA samples.
Related Concept Videos
03:00Next-generation Sequencing
The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.
01:54Base Excision Repair
One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
The first step of...
The first step of...
02:31Homologous Recombination
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
02:04Fixing Double-strand Breaks
The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...
01:57Sanger Sequencing
DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
02:25Overview of DNA Repair
In order to be passed through generations, genomic DNA must be undamaged and error-free. However, every day, DNA in a cell undergoes several thousand to a million damaging events by natural causes and external factors. Ionizing radiation such as UV rays, free radicals produced during cellular respiration, and hydrolytic damage from metabolic reactions can alter the structure of DNA. Damages caused include single-base alteration, base dimerization, chain breaks, and cross-linkage.
Chemically...
Chemically...