from the bench
Mycobacterium tuberculosis Next Generation Sequencing : Hitting the Easy Button
By Lucy DesJardin , PhD , director , Office of Research Development , State Hygienic Laboratory at the University of Iowa ; Valérie Reeb , PhD , clinical laboratory technical specialist , State Hygienic Laboratory at the University of Iowa ; Andrew Costello , clinical laboratory scientist , State Hygienic Laboratory at the University of Iowa ; and Ryan Jepson , M ( ASCP ), microbiology supervisor , State Hygienic Laboratory at the University of Iowa
The methods had different costs , ancillary instrumentation requirements , amounts of DNA recovered and hands-on time . However , there was no observed effect on NGS data quality relative to degree of genomic DNA fragmentation , presence of RNA in the extract or presence of contaminants as measured by the A260 / 280 or A260 / 230 ratios ( compromised to the greatest degree in InstaGene extracts ). This demonstrated that the library preparation step was quite robust and tolerant of fragmented input DNA and impurities .
Valérie Reeb prepares libraries for Mycobacterium tuberculosis whole-genome sequencing . Photo : IA SHL
Tuberculosis ( TB ) continues to be a major source of morbidity and mortality on a global scale . According to the US Centers for Disease Control and Prevention ( CDC ), 10.4 million people became sick with TB in 2016 , leading to 1.7 million TB-related deaths worldwide . TB is caused by Mycobacterium tuberculosis ( MTB ), which has several unusual characteristics that render it a public health threat . TB is spread through the air and requires a protracted monitoring period and extensive contact investigations to prevent the further spread of disease .
The US utilizes CDC ’ s TB Genotyping Information Management System ( TB GIMS ) for outbreak investigations that identify patient clusters . However , current MTB genotyping methods are not always sufficient to distinguish related from non-related cases . Whole genome sequencing ( WGS ) is an important new tool that provides additional information for genotyping and isolate characterization . Therefore , APHL and CDC ’ s Division of TB Elimination initiated a pilot program to enroll public health laboratories to perform WGS of MTB using next generation sequencing ( NGS ) technology .
An MTB Data Dive
NGS data must be of sufficient quality to provide good coverage across the entire genome for optimal genotyping and to identify point mutations in antibiotic resistance genes with high accuracy . MTB has a very dense cell wall that makes it difficult to extract DNA for NGS . It was noted that there were few studies directly comparing the time and cost involved with different MTB DNA isolation methods in relation to the quality of NGS data generated . It is understood that “ high quality DNA ” is needed , but specific parameters are not necessarily defined . Therefore , we performed a comparison of three nucleic acid isolation methods in order to evaluate the impact on DNA purity , library preparation , sequencing depth , uniformity of coverage , time and cost . The protocols compared were the CDC ’ s method using the Zymo Research fungal / bacterial DNA MicroPrep kit , Wadsworth Center ’ s Bio-Rad InstaGene matrix protocol and the MasterPure Complete DNA and RNA purification protocol from Epicenter .
MTB also grows very slowly and the shorter the time between isolation of MTB to WGS analysis , the faster the public health response . Typically , MTB isolates are grown in culture prior to sequencing . In order to shorten that step , we compared preparation of DNA from Mycobacterium Growth Indicator Tube cultures to extraction directly from frozen isolates that were shipped to the State Hygienic Laboratory from the CDC . We observed no impact on NGS data metrics . We were also able to prepare libraries from small amounts of bacteria , even when the bacterial pellet was not visible .
In summary , although the DNA quality was not optimal , the Bio-Rad InstaGene matrix protocol on frozen isolates ( no culture ) was the easiest method and provided quality NGS data at minimal cost and effort . The MasterPure preparation was lengthy , but yielded intact , high quality DNA , which could be important if performing long-read sequencing . Further studies could lead to a greater understanding of how the quantity and quality of microbial DNA affects NGS data analysis and help establish quality control guidelines . n
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18 LAB MATTERS Spring 2018
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