Metagenomic analysis of bacterial infections by means of high-throughput DNA sequencing

Abstract

DNA sequencing technology has undergone a remarkable and continuous improvement in recent years. The so-called 'high-throughput DNA sequencers' can determine hundreds of megabases of DNA sequences per run. We have been applying these new sequencers to the analysis of infectious diseases, especially bacterial infections. We review the efficacy of these sequencers, mainly based on our own experiences. The approach described here can be viewed as a metagenomic analysis of infectious diseases. The approach is in principle a method that does not depend on the type of target pathogens, so that it is possible to analyze various microorganisms, including bacteria, viruses, fungi and parasites, with a single common protocol. Applying this novel approach to cases in which infectious diseases are suspected because of environmental evidence, but the causative agent has not been identified, may lead to the discovery of unknown, novel pathogens. Also, the approach enables us to conduct an unbiased analysis of dynamics of the pathogen and associated microbiota in human specimens during the course of infectious diseases.

Pathogen-Specific Local Immune Fingerprints Diagnose Bacterial Infection in Peritoneal Dialysis Patients

“…Thus, for optimum stratification in future studies and to circumvent the limitations of conventional culture techniques, state-of-the-art molecular methods such as 16S and 23S rDNA amplification, mass spectrometry, and/or whole-sample sequencing and metagenomics might be utilized. [7][8][9][10][11]34,35 Such approaches, however, add a further level of complication in the analysis of patient results when the data produced are inconsistent or conflicting. Consequently, there is currently not sufficient evidence for recommending the routine use of molecular techniques for the diagnosis of peritonitis.…”

An ultra-deep sequencing strategy to detect sub-clonal TP53 mutations in presentation chronic lymphocytic leukaemia cases using multiple polymerases

“…Multiplexing using unique sample identifiers increases workload capacity and reduces cost. However, the sensitivity of deep sequencing also reports low frequency background errors generated by DNA polymerase during library preparation and the Illumina sequencing process itself [32][33][34] . Finding a way to distinguish low abundance mutants from systematic errors is therefore a key factor in the successful routine implementation of deep sequencing.…”

“…However, the depth of deep sequencing also leads to reporting of both systematic sequencing and amplification related errors, and distinguishing true variants from these events can be problematic [32][33][34] . Most bioinformatics approaches predict true variants from errors without taking the individual error rate at specific base positions along a sequence into consideration, with verification of low frequency events often limited to repetition of sequencing over two independent runs 15,16 .…”

Candidate gene discovery in autoimmunity by using extreme phenotypes, next generation sequencing and whole exome capture

“…Another problem observed with NGS is that it is prone to sequence specific errors that is a result of secondary structure formation on single stranded DNA molecules bound to the genome analyzer flow cell [19,20]. Examples of these include inverted repeats and GGC repetitive sequences.…”

“…As the strand continues to grow, the secondary structure's hydrogen bonds become destabilized and the reversible termination cycles for each sequence eventually resume at normal speed. However by this stage of the sequencing phase, it is likely that many reads proximal to the repeat region will falsely report a variant base due to the effects of lagged sequence contamination in previous cycles [19,20].…”