Profiling the BLAST bioinformatics application for load balancing on high-performance computing clusters.

BACKGROUND: The Basic Local Alignment Search Tool (BLAST) is a suite of commonly used algorithms for identifying matches between biological sequences. The user supplies a database file and query file of sequences for BLAST to find identical sequences between the two. The typical millions of database and query sequences make BLAST computationally challenging but also well suited for parallelization on high-performance computing clusters. The efficacy of parallelization depends on the data partitioning, where the optimal data partitioning relies on an accurate performance model. In previous studies, a BLAST job was sped up by 27 times by partitioning the database and query among thousands of processor nodes. However, the optimality of the partitioning method was not studied. Unlike BLAST performance models proposed in the literature that usually have problem size and hardware configuration as the only variables, the execution time of a BLAST job is a function of database size, query size, and hardware capability. In this work, the nucleotide BLAST application BLASTN was profiled using three methods: shell-level profiling with the Unix "time" command, code-level profiling with the built-in "profiler" module, and system-level profiling with the Unix "gprof" program. The runtimes were measured for six node types, using six different database files and 15 query files, on a heterogeneous HPC cluster with 500+ nodes. The empirical measurement data were fitted with quadratic functions to develop performance models that were used to guide the data parallelization for BLASTN jobs. RESULTS: Profiling results showed that BLASTN contains more than 34,500 different functions, but a single function, RunMTBySplitDB, takes 99.12% of the total runtime. Among its 53 child functions, five core functions were identified to make up 92.12% of the overall BLASTN runtime. Based on the performance models, static load balancing algorithms can be applied to the BLASTN input data to minimize the runtime of the longest job on an HPC cluster. Four test cases being run on homogeneous and heterogeneous clusters were tested. Experiment results showed that the runtime can be reduced by 81% on a homogeneous cluster and by 20% on a heterogeneous cluster by re-distributing the workload. DISCUSSION: Optimal data partitioning can improve BLASTN's overall runtime 5.4-fold in comparison with dividing the database and query into the same number of fragments. The proposed methodology can be used in the other applications in the BLAST+ suite or any other application as long as source code is available.

Epstein-Barr Virus in Burkitt Lymphoma in Africa Reveals a Limited Set of Whole Genome and LMP-1 Sequence Patterns: Analysis of Archival Datasets and Field Samples From Uganda, Tanzania, and Kenya.

Epstein-Barr virus (EBV) is associated with endemic Burkitt lymphoma (eBL), but the contribution of EBV variants is ill-defined. Studies of EBV whole genome sequences (WGS) have identified phylogroups that appear to be distinct for Asian versus non-Asian EBV, but samples from BL or Africa, where EBV was first discovered, are under-represented. We conducted a phylogenetic analysis of EBV WGS and LMP-1 sequences obtained primarily from BL patients in Africa and representative non-African EBV from other conditions or regions using data from GenBank, Sequence Read Archive, or Genomic Data Commons for the Burkitt Lymphoma Genome Sequencing Project (BLGSP) to generate data to support the use of a simpler biomarker of geographic or phenotypic associations. We also investigated LMP-1 patterns in 414 eBL cases and 414 geographically matched controls in the Epidemiology of Burkitt Lymphoma in East African children and minors (EMBLEM) study using LMP-1 PCR and Sanger sequencing. Phylogenetic analysis revealed distinct genetic patterns of African versus Asian EBV sequences. We identified 281 single nucleotide variations (SNVs) in LMP-1 promoter and coding region, which formed 12 unique patterns (A to L). Nine patterns (A, AB, C, D, F, I, J, K and L) predominated in African EBV, of which four were found in 92% of BL samples (A, AB, D, and H). Predominant patterns were B and G in Asia and H in Europe. EBV positivity in peripheral blood was detected in 95.6% of EMBLEM eBL cases versus 79.2% of the healthy controls (odds ratio [OR] =3.83; 95% confidence interval 2.06-7.14). LMP-1 was successfully sequenced in 66.7% of the EBV DNA positive cases but in 29.6% of the controls (ORs ranging 5-11 for different patterns). Four LMP-1 patterns (A, AB, D, and K) were detected in 63.1% of the cases versus 27.1% controls (ORs ranges: 5.58-11.4). Dual strain EBV infections were identified in WGS and PCR-Sanger data. In conclusion, EBV from Africa is phylogenetically separate from EBV in Asia. Genetic diversity in LMP-1 formed 12 patterns, which showed promising geographic and phenotypic associations. Presence of multiple strain infection should be considered in efforts to refine or improve EBV markers of ancestry or phenotype. Lay Summary: Epstein-Barr virus (EBV) infection, a ubiquitous infection, contributes to the etiology of both Burkitt Lymphoma (BL) and nasopharyngeal carcinoma, yet their global distributions vary geographically with no overlap. Genomic variation in EBV is suspected to play a role in the geographical patterns of these EBV-associated cancers, but relatively few EBV samples from BL have been comprehensively studied. We sought to compare phylogenetic patterns of EBV genomes obtained from BL samples in Africa and from tumor and non-tumor samples from elsewhere. We concluded that EBV obtained from BL in Africa is genetically separate from EBV in Asia. Through comprehensive analysis of nucleotide variations in EBV's LMP-1 gene, we describe 12 LMP-1 patterns, two of which (B and G) were found mostly in Asia. Four LMP-1 patterns (A, AB, D, and F) accounted for 92% of EBVs sequenced from BL in Africa. Our results identified extensive diversity of EBV, but BL in Africa was associated with a limited number of variants identified, which were different from those identified in Asia. Further research is needed to optimize the use of PCR and sequencing to study LMP-1 diversity for classification of EBV variants and for use in epidemiologic studies to characterize geographic and/or phenotypic associations of EBV variants with EBV-associated malignancies, including eBL.

Mitotic Errors Promote Genomic Instability and Leukemia in a Novel Mouse Model of Fanconi Anemia.

Fanconi anemia (FA) is a disease of genomic instability and cancer. In addition to DNA damage repair, FA pathway proteins are now known to be critical for maintaining faithful chromosome segregation during mitosis. While impaired DNA damage repair has been studied extensively in FA-associated carcinogenesis in vivo, the oncogenic contribution of mitotic abnormalities secondary to FA pathway deficiency remains incompletely understood. To examine the role of mitotic dysregulation in FA pathway deficient malignancies, we genetically exacerbated the baseline mitotic defect in Fancc-/- mice by introducing heterozygosity of the key spindle assembly checkpoint regulator Mad2. Fancc-/-;Mad2+/- mice were viable, but died from acute myeloid leukemia (AML), thus recapitulating the high risk of myeloid malignancies in FA patients better than Fancc-/-mice. We utilized hematopoietic stem cell transplantation to propagate Fancc-/-; Mad2+/- AML in irradiated healthy mice to model FANCC-deficient AMLs arising in the non-FA population. Compared to cells from Fancc-/- mice, those from Fancc-/-;Mad2+/- mice demonstrated an increase in mitotic errors but equivalent DNA cross-linker hypersensitivity, indicating that the cancer phenotype of Fancc-/-;Mad2+/- mice results from error-prone cell division and not exacerbation of the DNA damage repair defect. We found that FANCC enhances targeting of endogenous MAD2 to prometaphase kinetochores, suggesting a mechanism for how FANCC-dependent regulation of the spindle assembly checkpoint prevents chromosome mis-segregation. Whole-exome sequencing revealed similarities between human FA-associated myelodysplastic syndrome (MDS)/AML and the AML that developed in Fancc-/-; Mad2+/- mice. Together, these data illuminate the role of mitotic dysregulation in FA-pathway deficient malignancies in vivo, show how FANCC adjusts the spindle assembly checkpoint rheostat by regulating MAD2 kinetochore targeting in cell cycle-dependent manner, and establish two new mouse models for preclinical studies of AML.

[Decreasing the Incidence of Medical Adhesive-Related Skin Injuries in a Pediatric Intensive Care Unit].

BACKGROUND & PROBLEMS: Medical adhesives are typically used to fix wound dressings and catheters in place. Medical adhesive-related skin injuries (MARSI) are frequently caused by repetitive or improper usage of these products. The incidence rate in this unit is as high as 12.5%, which increases the difficulty and cost of care. After analysis of the situation, we identified the main causes of MARSI in our unit as: (1) Inadequate use of medical-adhesive products, (2) Lack of relevant education and training to prevent MARSI, and (3) lack of a standardized skin-damage-care procedure. PURPOSE: To decrease the incidence of MARSI in the pediatric intensive care unit. RESOLUTIONS: A training program was enacted to teach proper medical-adhesive application and removal techniques to caregivers. Consensus on care procedures was reached and care standards were modified. A mechanism for quality control was established. RESULTS: After implementing the program, the incidence of MARSI dropped from 12.5% to 5.18%, which achieved the target of this project. CONCLUSIONS: Other caregivers at our institution remain unaware of MARSI prevention techniques and protocols. We plan to continue cooperating with other staff members to prevent MARSI and to continue to reduce related skin injuries to as close to nil as possible.

Complete Genome Sequence of Feline Leukemia Virus Kawakami-Theilen Strain KT-FeLV-UCD-1.

This full-length genome sequence of a feline leukemia virus Kawakami-Theilen strain (designated KT-FeLV-UCD-1), produced from the chronically infected FL74-UCD-1 cell line, was obtained using high-throughput sequencing. It consisted of 8,464 bp and had a genomic organization similar to that of other gammaretroviruses, containing long terminal repeats and open reading frames for Gag, Pol, and Env.

Report of the 2019 NIST-FDA workshop on standards for next generation sequencing detection of viral adventitious agents in biologics and biomanufacturing.

Adventitious virus testing assures product safety by demonstrating the absence of viruses that could be unintentionally introduced during the manufacturing process. The capabilities of next-generation sequencing (NGS) for broad virus detection in biologics have been demonstrated by the detection of known and novel viruses that were previously missed using the recommended routine assays for adventitious agent testing. A meeting was co-organized by the National Institute of Standards and Technology and the U.S. Food and Drug Administration on September 18-19, 2019 in Gaithersburg, Maryland, USA, to facilitate standardization of NGS technologies for applications of adventitious virus testing in biologics. The goal was to assess the currently used standards for virus detection by NGS and their public availability, and to identify additional needs for different types of reference materials and standards (natural and synthetic). The meeting focused on the NGS processes from sample preparation through sequencing but did not thoroughly cover bioinformatics, since this was considered to be the topic of a separate meeting.

The Spodoptera frugiperda Sf9 cell line is a heterogeneous population of rhabdovirus-infected and virus-negative cells: Isolation and characterization of cell clones containing rhabdovirus X-gene variants and virus-negative cell clones.

The Sf9 cell line is broadly used for manufacturing baculovirus-expressed viral vaccines. We previously reported the presence of a novel, rhabdovirus in the Sf9 cell line, which contained a unique X gene (Sf-rhabdovirus; designated as X+ in this paper). These results were extended by other reports describing an Sf-rhabdovirus variant in Sf9 cells, which lacked 320 nucleotides encompassing the X-gene and adjacent intergenic region (designated as X- in this paper), and the development of an Sf-rhabdovirus negative cell line. Here, we report that the Sf9 cell line is a mixed-cell population, based upon isolation of cell clones with distinct phenotypes: Sf-rhabdovirus-negative, X+, and X-. We also show that Sf-rhabdovirus X+ and X- variants replicate independently in Sf-rhabdovirus-negative cells. These results shed light on the detection of different rhabdovirus variants by different laboratories using Sf9-derived cell clones and confirm that both X+ and X- viruses are infectious in rhabdovirus-negative Sf9 cells.

The macroH2A1.2 histone variant links ATRX loss to alternative telomere lengthening.

The growth of telomerase-deficient cancers depends on the alternative lengthening of telomeres (ALT), a homology-directed telomere-maintenance pathway. ALT telomeres exhibit a unique chromatin environment and generally lack the nucleosome remodeler ATRX, pointing to an epigenetic basis for ALT. Recently, we identified a protective role for the ATRX-interacting macroH2A1.2 histone variant during homologous recombination and replication stress (RS). Consistent with an inherent susceptibility to RS, we show that human ALT telomeres are highly enriched for macroH2A1.2. However, in contrast to ATRX-proficient cells, ALT telomeres transiently lose macroH2A1.2 during acute RS to facilitate DNA double-strand break (DSB) formation, a process that is almost completely prevented by ectopic ATRX expression. Telomeric macroH2A1.2 is re-deposited in a DNA damage response (DDR)-dependent manner to promote homologous recombination-associated ALT pathways. Our findings thus identify the dynamic exchange of macroH2A1.2 on chromatin as an epigenetic link among ATRX loss, RS-induced DDR initiation and telomere maintenance via homologous recombination.

Genomic Alterations of Staphylococcus aureus ATCC 25923 after Prolonged Passage in the Laboratory.

Staphylococcus aureus reference strain ATCC 25923 has been maintained for more than a decade in our laboratory. Genomic study revealed that the resulting strain AFIPCBER_B_8.4 has lost a 37-kb genomic fragment of the ATCC 25923 parental strain. The missing fragment showed sequence similarity to genes of bacteriophage proteins.

Comparative genomics, infectivity and cytopathogenicity of Zika viruses produced by acutely and persistently infected human hematopoietic cell lines.

Zika virus (ZIKV), an arthropod-borne virus, has emerged as a major human pathogen. Prolonged or persistent ZIKV infection of human cells and tissues may serve as a reservoir for the virus and present serious challenges to the safety of public health. Human hematopoietic cell lines with different developmental properties revealed differences in susceptibility and outcomes to ZIKV infection. In three separate studies involving the prototypic MR 766 ZIKV strain and the human monocytic leukemia U937 cell line, ZIKV initially developed only a low-grade infection at a slow rate. After continuous culture for several months, persistently ZIKV-infected cell lines were observed with most, if not all, cells testing positive for ZIKV antigen. The infected cultures produced ZIKV RNA (v-RNA) and infectious ZIKVs persistently ("persistent ZIKVs") with distinct infectivity and pathogenicity when tested using various kinds of host cells. When the genomes of ZIKVs from the three persistently infected cell lines were compared with the genome of the prototypic MR 766 ZIKV strain, distinct sets of mutations specific to each cell line were found. Significantly, all three "persistent ZIKVs" were capable of infecting fresh U937 cells with high efficiency at rapid rates, resulting in the development of a new set of persistently ZIKV-infected U937 cell lines. The genomes of ZIKVs from the new set of persistently ZIKV-infected U937 cell lines were further analyzed for their different mutations. The 2nd generation of persistent ZIKVs continued to possess most of the distinct sets of mutations specific to the respective 1st generation of persistent ZIKVs. We anticipate that the study will contribute to the understanding of the fundamental biology of adaptive mutations and selection during viral persistence. The persistently ZIKV-infected human cell lines that we developed will also be useful to investigate critical molecular pathways of ZIKV persistence and to study drugs or countermeasures against ZIKV infections and transmission.

Frequency of EBV LMP-1 Promoter and Coding Variations in Burkitt Lymphoma Samples in Africa and South America and Peripheral Blood in Uganda.

Epstein-Barr virus (EBV) is linked to several cancers, including endemic Burkitt lymphoma (eBL), but causal variants are unknown. We recently reported novel sequence variants in the LMP-1 gene and promoter in EBV genomes sequenced from 13 of 14 BL biopsies. Alignments of the novel sequence variants for 114 published EBV genomes, including 27 from BL cases, revealed four LMP-1 variant patterns, designated A to D. Pattern A variant was found in 48% of BL EBV genomes. Here, we used PCR-Sanger sequencing to evaluate 50 additional BL biopsies from Ghana, Brazil, and Argentina, and peripheral blood samples from 113 eBL cases and 115 controls in Uganda. Pattern A was found in 60.9% of 64 BL biopsies evaluated. Compared to PCR-negative subjects in Uganda, detection of Pattern A in peripheral blood was associated with eBL case status (odds ratio [OR] 31.7, 95% confidence interval: 6.8⁻149), controlling for relevant confounders. Variant Pattern A and Pattern D were associated with eBL case status, but with lower ORs (9.7 and 13.6, respectively). Our results support the hypothesis that EBV LMP-1 Pattern A may be associated with eBL, but it is not the sole associated variant. Further research is needed to replicate and elucidate our findings.

Lineage-specific SNPs for genotyping of Mycobacterium tuberculosis clinical isolates.

Tuberculosis (TB) is a severe infectious disease worldwide. Genetic variation of the causative agent, Mycobacterium tuberculosis (MTB), determines the outcomes of infection and anti-TB treatment. Until recently, there has been no effective and convenient way for classifying clinical isolates based on the DNA sequences of the divergent lineages of MTB infecting human populations. Here, we identified single nucleotide polymorphisms (SNPs) of six representative strains from Taiwan by whole-genome sequencing and comparing the results to the sequence of the H37Rv reference strain. One hundred and ten SNPs, each unique to one of the six strains, were used to genotype 150 additional isolates by applying DNA mass spectrometry. Lineage-specific SNPs were identified that could distinguish the major lineages of the clinical isolates. A subset including 32 SNPs was found to be sufficient to type four major groups of MTB isolates in Taiwan (ancient Beijing, modern Beijing, East African-Indian, and Latin-American Mediterranean). However, there was high genetic homozygosity within the Euro-American lineage, which included spoligotype-classified Haarlem and T strains. By whole-genome sequencing of 12 representative Euro-American isolates, we identified multiple subtype-specific SNPs which allowed us to distinguish two major branches within the Euro-American lineage.

Identification of Beijing lineage Mycobacterium tuberculosis with combined mycobacterial interspersed repetitive unit loci 26, 31, and ETR-A.

A rapid method for identification of Beijing lineage Mycobacterium tuberculosis is still needed in regions of tuberculosis endemicity, especially if genotyping methods are not readily accessible. After analyzing 1,557 clinical isolates, a PCR method with combined mycobacterial interspersed repetitive unit loci 26, 31, and ETR-A for differentiation of Beijing lineage isolates was established, the sensitivity and specificity of which are 94.7% and 98.5%, respectively.

A modified automated high-throughput mycobacterial interspersed repetitive unit method for genotyping Mycobacterium tuberculosis.

A total of 502 isolates of Mycobacterium tuberculosis collected in Taiwan were genotyped using a modified and high-throughput typing system involving mycobacterial interspersed repetitive units. The Hunter-Gaston discriminatory index improved significantly to 0.972 with the modified 15-locus scheme, including ETR-A, ETR-B, and ETR-C, compared with 0.951 for the previously reported 12-locus design.