Microbes and other organisms smaller than one to a few millimeters in size are hypothesized to have global populations, in contrast to the geographically restricted ranges of larger organisms. However, fungi, which routinely have reproductive propagules no larger than 10 micrometers, challenge the generality of this hypothesis because recent studies have shown that globally distributed morphological species embrace two or more geographically restricted phylogenetic species. We used the concordance of gene genealogies to recognize phylogenetic species in the globally distributed opportunistic human pathogenic fungus, Aspergillus fumigatus. Based on DNA sequence data of five loci for each of 63 individuals collected from five continents, we have delineated two phylogenetic species in this single morphological species. Unlike all other fungi examined to date, both genetically isolated groups showed a global distribution with no evidence of a correlation between genotype and geographic location. Sexual reproduction has never been observed in A. fumigatus, but when the same data were used to explore the association of alleles at the five loci for one of the phylogenetic species, evidence was found to support recombination. The discovery of a cryptic species is medically relevant because different species are likely to differ in virulence or drug resistance. The discovery of a globally distributed A. fumigatus species clade highlights the need for ecological studies of the fungus to either document global dispersal or propose alternative mechanisms by which it persists as single, global phylogenetic population.

Abstract. Microbes and other organisms smaller than one to a few millimeters in size are hypothesized to have global populations, in contrast to the geographically restricted ranges of larger organisms. However, fungi, which routinely have reproductive propagules no larger than 10 micrometers, challenge the generality of this hypothesis because recent studies have shown that globally distributed morphological species embrace two or more geographically restricted phylogenetic species. We used the concordance of gene genealogies to recognize phylogenetic species in the globally distributed opportunistic human pathogenic fungus, Aspergillus fumigatus. Based on DNA sequence data of five loci for each of 63 individuals collected from five continents, we have delineated two phylogenetic species in this single morphological species. Unlike all other fungi examined to date, both genetically isolated groups showed a global distribution with no evidence of a correlation between genotype and geographic location. Sexual reproduction has never been observed in A. fumigatus, but when the same data were used to explore the association of alleles at the five loci for one of the phylogenetic species, evidence was found to support recombination. The discovery of a cryptic species is medically relevant because different species are likely to differ in virulence or drug resistance. The discovery of a globally distributed A. fumigatus species clade highlights the need for ecological studies of the fungus to either document global dispersal or propose alternative mechanisms by which it persists as single, global phylogenetic population.

Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.

A tropism test is required prior to initiation of CCR5 antagonist therapy in HIV-1 infected individuals, as these agents are not effective in patients harboring CXCR4 (X4) coreceptor-using viral variants. We developed a clinical laboratory-based genotypic tropism test for detection of CCR5-using (R5) or X4 variants that utilizes triplicate population sequencing (TPS) followed by ultradeep sequencing (UDS) for samples classified as R5. Tropism was inferred using the bioinformatic algorithms geno2pheno[coreceptor] and PSSMx4r5. Virologic response as a function of tropism readout was retrospectively assessed using blinded samples from treatment-experienced subjects who received maraviroc (N = 327) in the MOTIVATE and A4001029 clinical trials. MOTIVATE patients were classified as R5 and A4001029 patients were classified as non-R5 by the original Trofile test. Virologic response was compared between the R5 and non-R5 groups determined by TPS, UDS alone, the reflex strategy and the Trofile Enhanced Sensitivity (TF-ES) test. UDS had greater sensitivity than TPS to detect minority non-R5 variants. The median log10 viral load change at week 8 was −2.4 for R5 subjects, regardless of the method used for classification; for subjects with non-R5 virus, median changes were −1.2 for TF-ES or the Reflex Test and −1.0 for UDS. The differences between R5 and non-R5 groups were highly significant in all 3 cases (p<0.0001). At week 8, the positive predictive value was 66% for TF-ES and 65% for both the Reflex test and UDS. Negative predictive values were 59% for TF-ES, 58% for the Reflex Test and 61% for UDS. In conclusion, genotypic tropism testing using UDS alone or a reflex strategy separated maraviroc responders and non-responders as well as a sensitive phenotypic test, and both assays showed improved performance compared to TPS alone. Genotypic tropism tests may provide an alternative to phenotypic testing with similar discriminating ability.

The objective of this study was to design and validate a next-generation sequencing assay (NGS) to detect BRCA1 and BRCA2 mutations. We developed an assay using random shearing of genomic DNA followed by RNA bait tile hybridization and NGS sequencing on both the Illumina MiSeq and Ion Personal Gene Machine (PGM). We determined that the MiSeq Reporter software supplied with the instrument could not detect deletions greater than 9 base pairs. Therefore, we developed an alternative alignment and variant calling software, Quest Sequencing Analysis Pipeline (QSAP), that was capable of detecting large deletions and insertions. In validation studies, we used DNA from 27 stem cell lines, all with known deleterious BRCA1 or BRCA2 mutations, and DNA from 67 consented control individuals who had a total of 352 benign variants. Both the MiSeq/QSAP combination and PGM/Torrent Suite combination had 100% sensitivity for the 379 known variants in the validation series. However, the PGM/Torrent Suite combination had a lower intra- and inter-assay precision of 96.2% and 96.7%, respectively when compared to the MiSeq/QSAP combination of 100% and 99.4%, respectively. All PGM/Torrent Suite inconsistencies were false-positive variant assignments. We began commercial testing using both platforms and in the first 521 clinical samples MiSeq/QSAP had 100% sensitivity for BRCA1/2 variants, including a 64-bp deletion and a 10-bp insertion not identified by PGM/Torrent Suite, which also suffered from a high false-positive rate. Neither the MiSeq nor PGM platform with their supplied alignment and variant calling software are appropriate for a clinical laboratory BRCA sequencing test. We have developed an NGS BRCA1/2 sequencing assay, MiSeq/QSAP, with 100% analytic sensitivity and specificity in the validation set consisting of 379 variants. The MiSeq/QSAP combination has sufficient performance for use in a clinical laboratory.