Non-biological synthetic spike-in controls and the AMPtk software pipeline improve mycobiome data

High-throughput amplicon sequencing (HTAS) of conserved DNA regions is a powerful technique to characterize microbial communities. Recently, spike-in mock communities have been used to measure accuracy of sequencing platforms and data analysis pipelines. To assess the ability of sequencing platforms and data processing pipelines using fungal internal transcribed spacer (ITS) amplicons, we created two ITS spike-in control mock communities composed of cloned DNA in plasmids: a biological mock community, consisting of ITS sequences from fungal taxa, and a synthetic mock community (SynMock), consisting of non-biological ITS-like sequences. Using these spike-in controls we show that: (1) a non-biological synthetic control (e.g., SynMock) is the best solution for parameterizing bioinformatics pipelines, (2) pre-clustering steps for variable length amplicons are critically important, (3) a major source of bias is attributed to the initial polymerase chain reaction (PCR) and thus HTAS read abundances are typically not representative of starting values. We developed AMPtk, a versatile software solution equipped to deal with variable length amplicons and quality filter HTAS data based on spike-in controls. While we describe herein a non-biological SynMock community for ITS sequences, the concept and AMPtk software can be widely applied to any HTAS dataset to improve data quality.

Use of Pyrosequencing to Quantify Incidence of a Specific Aspergillus flavus Strain Within Complex Fungal Communities Associated with Commercial Cotton Crops

Atoxigenic strains of Aspergillus flavus have been used as aflatoxin management tools on over 50,000 hectares of commercial crops since 2000. To assess treatment efficacy, atoxigenic strain incidence is routinely monitored by vegetative compatibility analyses (VCA) that require culturing, generation of auxotrophs, and complementation with tester mutants. Two pyrosequencing assays (PA) that require no culturing were developed for monitoring incidences of atoxigenic strains on ginned cottonseed. The assays, which quantify frequencies of characteristic single nucleotide polymorphisms (SNPs) in the aflR and pksA genes, were validated against standard VCA on cottonseed collected from commercial gins in South Texas, Arizona, and Southern California where the atoxigenic strain AF36 is used to manage aflatoxin contamination. Cottonseed washings were subjected to both VCA and PA. PA was performed directly on DNA isolated from particulates pelleted from the wash water by centrifugation. Addition of CaCl2 and diatomaceous earth prior to pelleting increased the amount of DNA isolated. Accuracy and reproducibility of the PA were contrasted with those for the VCA that has been used for over a decade. Correlation coefficients between VCA and PA indicated good correspondence between the results from the two assays (r = 0.91 for aflR assay and r = 0.80 for pksA assay). PAs were highly variable for samples with low incidences of A. flavus due to variability in the initial polymerase chain reaction step. This held for both DNA isolated from cottonseed washes and for mixtures of purified DNA. For samples yielding low quantities of A. flavus DNA, averaging of results from 4 to 5 replicates was required to achieve acceptable correlations with VCA. Pyrosequencing has the potential to become a powerful tool for monitoring atoxigenic strains within complex A. flavus communities without limitations imposed by traditional culturing methods.

Possible transmission of SARS within the United Kingdom

The sixth probable case of sudden acute respiratory syndrome (SARS) diagnosed in the United Kingdom (UK) was reported to the World Health Organization (WHO) on 11 April (1). The patient has been isolated in hospital where his condition is reported as stable. He was diagnosed as a probable SARS case because of having a significant respiratory illness with radiological signs, and having had close contact with a probable case of SARS. Laboratory investigations are underway at the Central Public Health Laboratory of the Health Protection Agency. Initial tests for coronavirus have been negative. It is, however, recognized that such initial (polymerase chain reaction (PCR)) tests can be negative in a person who is infected with the SARS virus. Definitive results through antibody testing of acute and convalescent sera will be available later (2, 3).

A molecular protocol for diagnosing myotonic dystrophy

Myotonic dystrophy (DM) is an autosomal dominant genetic disease caused by an unstable CTG repeat sequence in the 3' untranslated region of the myotonin protein kinase gene. The CTG repeat is present 5-30 times in the normal population, whereas DM patients have CTG expansions of 50 to several thousand repeats. The age of onset of the disorder and the severity of the phenotype is roughly correlated with the size of the CTG expansion. We developed a molecular protocol for the diagnosis of DM based on an initial polymerase chain reaction screen to detect normal-sized alleles and small expansions, followed by an improved Southern protocol to detect larger expansions.