skip to main content

DOE PAGESDOE PAGES

Title: A systematic comparison of error correction enzymes by next-generation sequencing

Gene synthesis, the process of assembling genelength fragments from shorter groups of oligonucleotides (oligos), is becoming an increasingly important tool in molecular and synthetic biology. The length, quality and cost of gene synthesis are limited by errors produced during oligo synthesis and subsequent assembly. Enzymatic error correction methods are cost-effective means to ameliorate errors in gene synthesis. Previous analyses of these methods relied on cloning and Sanger sequencing to evaluate their efficiencies, limiting quantitative assessment. Here, we develop a method to quantify errors in synthetic DNA by next-generation sequencing. We analyzed errors in model gene assemblies and systematically compared six different error correction enzymes across 11 conditions. We find that ErrASE and T7 Endonuclease I are the most effective at decreasing average error rates (up to 5.8-fold relative to the input), whereas MutS is the best for increasing the number of perfect assemblies (up to 25.2-fold). We are able to quantify differential specificities such as ErrASE preferentially corrects C/G transversions whereas T7 Endonuclease I preferentially corrects A/T transversions. More generally, this experimental and computational pipeline is a fast, scalable and extensible way to analyze errors in gene assemblies, to profile error correction methods, and to benchmark DNA synthesis methods.
Authors:
ORCiD logo [1] ;  [2] ;  [3] ;  [4] ; ORCiD logo [1]
  1. Univ. of California, Los Angeles, CA (United States); UCLA-DOE Inst. for Genomics and Proteomics, Los Angeles, CA (United States)
  2. Univ. of Pennsylvania, Philadelphia, PA (United States)
  3. Univ. of California, Los Angeles, CA (United States)
  4. Brigham and Women's Hospital (Harvard Medical School), Boston, MA (United States); Wyss Inst. for Biologically Inspired Engineering, Boston, MA (United States)
Publication Date:
Grant/Contract Number:
FC02-02ER63421
Type:
Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 45; Journal Issue: 15; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Research Org:
Univ. of California, Los Angeles, CA (United States)
Sponsoring Org:
USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES
OSTI Identifier:
1425978