title: Comparison of Sample Preparation Methods Used for the Next-Generation Sequencing of Mycobacterium tuberculosis.
abstract: The advent and widespread application of next-generation sequencing (NGS) technologies to the study of microbial genomes has led to a substantial increase in the number of studies in which whole genome sequencing (WGS) is applied to the analysis of microbial genomic epidemiology. However, microorganisms such as Mycobacterium tuberculosis (MTB) present unique problems for sequencing and downstream analysis based on their unique physiology and the composition of their genomes. In this study, we compare the quality of sequence data generated using the Nextera and TruSeq isolate preparation kits for library construction prior to Illumina sequencing-by-synthesis. Our results confirm that MTB NGS data quality is highly dependent on the purity of the DNA sample submitted for sequencing and its guanine-cytosine content (or GC-content). Our data additionally demonstrate that the choice of library preparation method plays an important role in mitigating downstream sequencing quality issues. Importantly for MTB, the Illumina TruSeq library preparation kit produces more uniform data quality than the Nextera XT method, regardless of the quality of the input DNA. Furthermore, specific genomic sequence motifs are commonly missed by the Nextera XT method, as are regions of especially high GC-content relative to the rest of the MTB genome. As coverage bias is highly undesirable, this study illustrates the importance of appropriate protocol selection when performing NGS studies in order to ensure that sound inferences can be made regarding mycobacterial genomes.
authors: National Microbiology Laboratory, National Reference Centre for Mycobacteriology, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors: Andrea D
authors: AD
authors: Tyler
authors: National Microbiology Laboratory, National Reference Centre for Mycobacteriology, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors: Sara
authors: S
authors: Christianson
authors: Science Technology Cores & Services Division, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors: Natalie C
authors: NC
authors: Knox
authors: Science Technology Cores & Services Division, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors: Philip
authors: P
authors: Mabon
authors: National Microbiology Laboratory, National Reference Centre for Mycobacteriology, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors: Joyce
authors: J
authors: Wolfe
authors: Science Technology Cores & Services Division, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.|Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.|Department of Computer Science, University of Manitoba, Winnipeg, Manitoba, Canada.
authors: Gary
authors: G
authors: Van Domselaar
authors: Science Technology Cores & Services Division, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.|Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
authors: Morag R
authors: MR
authors: Graham
authors: National Microbiology Laboratory, National Reference Centre for Mycobacteriology, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.|Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
authors: Meenu K
authors: MK
authors: Sharma
mesh_terms: D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification; D013048:Specimen Handling / Q000379:methods*