Parsnp 2.0: scalable core-genome alignment for massive microbial datasets.

MOTIVATION: Since 2016, the number of microbial species with available reference genomes in NCBI has more than tripled. Multiple genome alignment, the process of identifying nucleotides across multiple genomes which share a common ancestor, is used as the input to numerous downstream comparative analysis methods. Parsnp is one of the few multiple genome alignment methods able to scale to the current era of genomic data; however, there has been no major release since its initial release in 2014. RESULTS: To address this gap, we developed Parsnp v2, which significantly improves on its original release. Parsnp v2 provides users with more control over executions of the program, allowing Parsnp to be better tailored for different use-cases. We introduce a partitioning option to Parsnp, which allows the input to be broken up into multiple parallel alignment processes which are then combined into a final alignment. The partitioning option can reduce memory usage by over 4× and reduce runtime by over 2×, all while maintaining a precise core-genome alignment. The partitioning workflow is also less susceptible to complications caused by assembly artifacts and minor variation, as alignment anchors only need to be conserved within their partition and not across the entire input set. We highlight the performance on datasets involving thousands of bacterial and viral genomes. AVAILABILITY AND IMPLEMENTATION: Parsnp v2 is available at https://github.com/marbl/parsnp.

Parsnp 2.0: Scalable Core-Genome Alignment for Massive Microbial Datasets.

Motivation: Since 2016, the number of microbial species with available reference genomes in NCBI has more than tripled. Multiple genome alignment, the process of identifying nucleotides across multiple genomes which share a common ancestor, is used as the input to numerous downstream comparative analysis methods. Parsnp is one of the few multiple genome alignment methods able to scale to the current era of genomic data; however, there has been no major release since its initial release in 2014. Results: To address this gap, we developed Parsnp v2, which significantly improves on its original release. Parsnp v2 provides users with more control over executions of the program, allowing Parsnp to be better tailored for different use-cases. We introduce a partitioning option to Parsnp, which allows the input to be broken up into multiple parallel alignment processes which are then combined into a final alignment. The partitioning option can reduce memory usage by over 4x and reduce runtime by over 2x, all while maintaining a precise core-genome alignment. The partitioning workflow is also less susceptible to complications caused by assembly artifacts and minor variation, as alignment anchors only need to be conserved within their partition and not across the entire input set. We highlight the performance on datasets involving thousands of bacterial and viral genomes. Availability: Parsnp is available at https://github.com/marbl/parsnp.

Olivar: towards automated variant aware primer design for multiplex tiled amplicon sequencing of pathogens.

Tiled amplicon sequencing has served as an essential tool for tracking the spread and evolution of pathogens. Over 15 million complete SARS-CoV-2 genomes are now publicly available, most sequenced and assembled via tiled amplicon sequencing. While computational tools for tiled amplicon design exist, they require downstream manual optimization both computationally and experimentally, which is slow and costly. Here we present Olivar, a first step towards a fully automated, variant-aware design of tiled amplicons for pathogen genomes. Olivar converts each nucleotide of the target genome into a numeric risk score, capturing undesired sequence features that should be avoided. In a direct comparison with PrimalScheme, we show that Olivar has fewer mismatches overlapping with primers and predicted PCR byproducts. We also compare Olivar head-to-head with ARTIC v4.1, the most widely used primer set for SARS-CoV-2 sequencing, and show Olivar yields similar read mapping rates (~90%) and better coverage to the manually designed ARTIC v4.1 amplicons. We also evaluate Olivar on real wastewater samples and found that Olivar has up to 3-fold higher mapping rates while retaining similar coverage. In summary, Olivar automates and accelerates the generation of tiled amplicons, even in situations of high mutation frequency and/or density. Olivar is available online as a web application at https://olivar.rice.edu  and can be installed locally as a command line tool with Bioconda. Source code, installation guide, and usage are available at https://github.com/treangenlab/Olivar .

Olivar: automated variant aware primer design for multiplex tiled amplicon sequencing of pathogens.

Tiled amplicon sequencing has served as an essential tool for tracking the spread and evolution of pathogens. Over 2 million complete SARS-CoV-2 genomes are now publicly available, most sequenced and assembled via tiled amplicon sequencing. While computational tools for tiled amplicon design exist, they require downstream manual optimization both computationally and experimentally, which is slow and costly. Here we present Olivar, a first step towards a fully automated, variant-aware design of tiled amplicons for pathogen genomes. Olivar converts each nucleotide of the target genome into a numeric risk score, capturing undesired sequence features that should be avoided. In a direct comparison with PrimalScheme, we show that Olivar has fewer SNPs overlapping with primers and predicted PCR byproducts. We also compared Olivar head-to-head with ARTIC v4.1, the most widely used primer set for SARS-CoV-2 sequencing, and show Olivar yields similar read mapping rates (~90%) and better coverage to the manually designed ARTIC v4.1 amplicons. We also evaluated Olivar on real wastewater samples and found that Olivar had up to 3-fold higher mapping rates while retaining similar coverage. In summary, Olivar automates and accelerates the generation of tiled amplicons, even in situations of high mutation frequency and/or density. Olivar is available as a web application at https://olivar.rice.edu. Olivar can also be installed locally as a command line tool with Bioconda. Source code, installation guide and usage are available at https://github.com/treangenlab/Olivar.

Electromagnetic controlled cortical impact device for precise, graded experimental traumatic brain injury.

Genetically modified mice represent useful tools for traumatic brain injury (TBI) research and attractive preclinical models for the development of novel therapeutics. Experimental methods that minimize the number of mice needed may increase the pace of discovery. With this in mind, we developed and characterized a prototype electromagnetic (EM) controlled cortical impact device along with refined surgical and behavioral testing techniques. By varying the depth of impact between 1.0 and 3.0 mm, we found that the EM device was capable of producing a broad range of injury severities. Histologically, 2.0-mm impact depth injuries produced by the EM device were similar to 1.0-mm impact depth injuries produced by a commercially available pneumatic device. Behaviorally, 2.0-, 2.5-, and 3.0-mm impacts impaired hidden platform and probe trial water maze performance, whereas 1.5-mm impacts did not. Rotorod and visible platform water maze deficits were also found following 2.5- and 3.0-mm impacts. No impairment of conditioned fear performance was detected. No differences were found between sexes of mice. Inter-operator reliability was very good. Behaviorally, we found that we could statistically distinguish between injury depths differing by 0.5 mm using 12 mice per group and between injury depths differing by 1.0 mm with 7-8 mice per group. Thus, the EM impactor and refined surgical and behavioral testing techniques may offer a reliable and convenient framework for preclinical TBI research involving mice.

Number of lymph nodes examined and its impact on colorectal cancer staging.

The status of lymph nodes is the most important prognosticator in colorectal cancer patients. Patients with lymph node involvement have a lower survival rate and are candidates for adjuvant therapy. The purpose of our study was to determine the number of lymph nodes that needs to be examined to accurately detect nodal metastasis. We conducted a retrospective study of 151 patients who underwent colorectal cancer operation at Harbor-UCLA Medical Center. Data from the operative report and pathology report were collected and analyzed. Fourteen (33.3%) patients with five to nine nodes examined had positive nodes. Twenty-six (57.8%) patients with 10 to 14 nodes examined had positive nodes. Patients who had 10 to 14 nodes examined were significantly more likely to have positive lymph nodes (P = 0.03). Patients with advanced T stage had a significantly higher number of positive lymph nodes (78.1% in T4 vs 11.1% in T1, P < 0.0001). Patients with poorly differentiated cancer showed a trend toward a higher positive node rate. Tumor differentiation and T stage seem to correlate with higher nodal metastasis rate. A higher number of lymph nodes examined was associated with a higher nodal metastasis rate. Examination of at least 10 lymph nodes would increase the yield of positive lymph nodes and avoid under-staging of patients with colorectal cancer.

Human apolipoprotein E4 worsens acute axonal pathology but not amyloid-ß immunoreactivity after traumatic brain injury in 3xTG-AD mice.

Apolipoprotein E4 (APOE4) genotype is a risk factor for poor outcome after traumatic brain injury (TBI), particularly in young patients, but the underlying mechanisms are not known. By analogy to effects of APOE4 on the risk of Alzheimer disease (AD), the APOE genotype may influence ß-amyloid (Aß) and tau deposition after TBI. To test this hypothesis, we crossed 3xTG-AD transgenic mice carrying 3 human familial AD mutations (PS1(M146V), tauP(301)L, and APP(SWE)) to human ApoE2-, ApoE3-, and ApoE4-targeted replacement mice. Six- to 8-month-old 3xTG-ApoE mice were assayed by quantitative immunohistochemistry for amyloid precursor protein (APP), Aß(1-40) (Aß40), Aß(1-42) (Aß42), total human tau, and phospho-serine 199 (pS199) tau at 24 hours after moderate controlled cortical impact. There were increased numbers of APP-immunoreactive axonal varicosities in 3xTG-ApoE4 mice versus the other genotypes. This finding was repeated in a separate cohort of ApoE4-targeted replacement mice without human transgenes compared with ApoE3 and ApoE2 mice. There were no differences between genotypes in the extent of intra-axonal Aß40 and Aß42; none of the mice had extracellular Aß deposition. Regardless of injury status, 3xTG-ApoE4 mice had more total human tau accumulation in both somatodendritic and intra-axonal compartments than other genotypes. These results suggest that the APOE4 genotype may have a primary effect on the severity of axonal injury in acute TBI.