Estrategias para rentabilizar un establo lechero de la región Arequipa

La presente tesis desarrolla un modelo de gestión estratégica utilizando la herramienta del Balanced Scorecard para la empresa “Establo Víctor Manuel” de la región Arequipa, con el objetivo de formular y seleccionar las estrategias más adecuadas que permitan aumentar la rentabilidad de la empresa, ya que anteriormente no se ha desarrollado ningún plan estratégico. La empresa “Establo Víctor Manuel” se encuentra ubicada en la ciudad de Arequipa y es uno de los proveedores de leche fresca más importantes de la región Arequipa de la empresa GRUPO GLORIA; la calidad de la leche fresca producida por la empresa es reconocida por su cliente como una de las mejores de la región. Para llevar a cabo la elaboración de la propuesta, se inició haciendo un análisis situacional actual de la empresa, ello apoyado con el análisis interno y externo, con la finalidad de determinar la influencia del entorno en la empresa y conocer las fuerzas claves que afectan su desempeño. Una vez realizado dicho análisis, se pasó a conformar la propuesta, realizando las declaratorias de la misión, visión y valores de la empresa, pasando después a plantear los objetivos estratégicos con el apoyo de las técnicas analíticas, matrices, que nos permitirán identificar todas las estrategias resultantes, luego de ello se hará una selección definitiva de estrategias. Para terminar la planificación estratégica, se procede a diseñar el mapa estratégico y el cuadro de mando integral. Finalmente se concluye que los principales objetivos estratégicos son: incrementar la rentabilidad, reducir los costos y asegurar la calidad del producto, para ello, se definieron iniciativas estratégicas, indicadores y metas

Investigating the Extent of Primer Dropout in SARS-CoV-2 Genome Sequences During the Early Circulation of Delta Variants

The SARS-CoV-2 Delta variant, corresponding to the Pangolin lineage B.1.617.2, was first detected in India in July 2020 and rapidly became dominant worldwide. The ARTIC v3 protocol for SARS-CoV-2 whole-genome sequencing, which relies on a large number of PCR primers, was among the first available early in the pandemic, but may be prone to coverage dropouts that result in incomplete genome sequences. A new set of primers (v4) was designed to circumvent this issue in June 2021. In this study, we investigated whether the sequencing community adopted the new sets of primers, especially in the context of the spread of the Delta lineage, in July 2021. Because information about protocols from individual laboratories is generally difficult to obtain, the aims of the study were to identify whether large under-sequenced regions were present in deposited Delta variant genome sequences (from April to August 2021), to investigate the extent of the coverage dropout among all the currently available Delta sequences in six countries, and to propose simple PCR primer modifications to sequence the missing region, especially for the first circulating Delta variants observed in 2021 in Switzerland. Candidate primers were tested on few clinical samples, highlighting the need to further pursue primer optimization and validation on a larger and diverse set of samples

Evaluation of whole- genome sequence data analysis approaches for short- and long- read sequencing of Mycobacterium tuberculosis

Whole-genome sequencing (WGS) of Mycobacterium tuberculosis (MTB) isolates can be used to get an accurate diagnosis, to guide clinical decision making, to control tuberculosis (TB) and for outbreak investigations. We evaluated the performance of long-read (LR) and/or short-read (SR) sequencing for anti-TB drug-resistance prediction using the TBProfiler and Mykrobe tools, the fraction of genome recovery, assembly accuracies and the robustness of two typing approaches based on core-genome SNP (cgSNP) typing and core-genome multi-locus sequence typing (cgMLST). Most of the discrepancies between phenotypic drug-susceptibility testing (DST) and drug-resistance prediction were observed for the first-line drugs rifampicin, isoniazid, pyrazinamide and ethambutol, mainly with LR sequence data. Resistance prediction to second-line drugs made by both TBProfiler and Mykrobe tools with SR- and LR-sequence data were in complete agreement with phenotypic DST except for one isolate. The SR assemblies were more accurate than the LR assemblies, having significantly (P<0.05) fewer indels and mismatches per 100 kbp. However, the hybrid and LR assemblies had slightly higher genome fractions. For LR assemblies, Canu followed by Racon, and Medaka polishing was the most accurate approach. The cgSNP approach, based on either reads or assemblies, was more robust than the cgMLST approach, especially for LR sequence data. In conclusion, anti-TB drug-resistance prediction, particularly with only LR sequence data, remains challenging, especially for first-line drugs. In addition, SR assemblies appear more accurate than LR ones, and reproducible phylogeny can be achieved using cgSNP approaches

NASCarD (Nanopore Adaptive Sampling with Carrier DNA): A rapid, PCR-free method for whole genome sequencing of pathogens in clinical samples

Whole-genome sequencing (WGS) represents the main technology for SARS-CoV-2 lineage characterization in diagnostic laboratories worldwide. The rapid, near-full-length sequencing of the viral genome is commonly enabled by high-throughput sequencing of PCR amplicons derived from cDNA molecules. Here, we present a new approach, called NASCarD (Nanopore adaptive sampling with carrier DNA), which allows low amount of nucleic acids to be sequenced while selectively enriching for sequences of interest, hence limiting the production of non-target sequences. Using clinical samples positive for SARS-CoV-2 during the Omicron wave, we demonstrate how the method leads to up to &gt;100x coverage of the full genome sequences of the target organism as compared to standard shotgun metatranscriptomics approach. It provides complete and accurate genome sequence reconstruction within seven hours at a competitive cost. The new approach may have applications beyond SARS-CoV-2 sequencing for other DNA or RNA pathogens in clinical samples

NASCarD (Nanopore Adaptive Sampling with Carrier DNA): A Rapid, PCR-Free Method for SARS-CoV-2 Whole-Genome Sequencing in Clinical Samples.

Whole-genome sequencing (WGS) represents the main technology for SARS-CoV-2 lineage characterization in diagnostic laboratories worldwide. The rapid, near-full-length sequencing of the viral genome is commonly enabled by high-throughput sequencing of PCR amplicons derived from cDNA molecules. Here, we present a new approach called NASCarD (Nanopore Adaptive Sampling with Carrier DNA), which allows a low amount of nucleic acids to be sequenced while selectively enriching for sequences of interest, hence limiting the production of non-target sequences. Using COVID-19 positive samples available during the omicron wave, we demonstrate how the method may lead to >99% genome completeness of the SARS-CoV-2 genome sequences within 7 h of sequencing at a competitive cost. The new approach may have applications beyond SARS-CoV-2 sequencing for other DNA or RNA pathogens in clinical samples

Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing.

Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains

Investigating the biological and technical origins of unknown bases in the S region of the SARS-CoV-2 Delta variant genome sequences

We are reporting on the observation of a large, under-sequenced region of the S gene of the SARS-CoV2 Delta variant genomes, identified in sequences originating from various sequencing centres worldwide (e.g. USA, India, England, Switzerland, France, Germany). This poorly sequenced region was identified from the early phases of the Delta variant spread and the phenomenon is still ongoing. As many commonly-used protocols rely on amplicon-based sequencing procedures, we investigated the likely origin of the issue. We established its biological origin as resulting from mutations in the viral genomes at primer binding sites. We designed and evaluated new PCR primers to circumvent this issue in order to complement the ARTIC v3 set, and validated their performance for the sequencing of circulating Delta variants

A Sample-to-Report Solution for Taxonomic Identification of Cultured Bacteria in the Clinical Setting Based on Nanopore Sequencing.

Amplicon sequencing of 16S rRNA gene is commonly used for the identification of bacterial isolates in diagnostic laboratories, and mostly relies on the Sanger sequencing method. The latter, however, suffers from a number of limitations with the most significant being the inability to resolve mixed amplicons when closely related species are co-amplified from a mixed culture. This often leads to either increased turnaround time or absence of usable sequence data. Short-read NGS technologies could solve the mixed amplicon issue, but would lack both cost efficiency at low throughput and fast turnaround times. Nanopore sequencing developed by Oxford Nanopore Technologies (ONT) could solve those issues by enabling flexible number of samples per run and adjustable sequencing time. Here we report on the development of a standardized laboratory workflow combined with a fully automated analysis pipeline LORCAN (Long Read Consensus ANalysis), which together provide a sample-to-report solution for amplicon sequencing and taxonomic identification of the resulting consensus sequences. Validation of the approach was conducted on a panel of reference strains and on clinical samples consisting of single or mixed rRNA amplicons associated with various bacterial genera by direct comparison to the corresponding Sanger sequences. Additionally, simulated read and amplicon mixtures were used to assess LORCAN's behaviour when dealing with samples with known cross-contamination level. We demonstrate that by combining ONT amplicon sequencing results with LORCAN, the accuracy of Sanger sequencing can be closely matched (>99.6% sequence identity) and that mixed samples can be resolved at the single base resolution level. The presented approach has the potential to significantly improve the flexibility, reliability and availability of amplicon sequencing in diagnostic settings

Rapid and Cost-Efficient Enterovirus Genotyping from Clinical Samples Using Flongle Flow Cells.

Enteroviruses affect millions of people worldwide and are of significant clinical importance. The standard method for enterovirus identification and genotyping still relies on Sanger sequencing of short diagnostic amplicons. In this study, we assessed the feasibility of nanopore sequencing using the new flow cell "Flongle" for fast, cost-effective, and accurate genotyping of human enteroviruses from clinical samples. PCR amplification of partial VP1 gene was performed from multiple patient samples, which were multiplexed together after barcoding PCR and sequenced multiple times on Flongle flow cells. The nanopore consensus sequences obtained from mapping reads to a reference database were compared to their Sanger sequence counterparts. Using clinical specimens sampled over different years, we were able to correctly identify enterovirus species and genotypes for all tested samples, even when doubling the number of barcoded samples on one flow cell. Average sequence identity across sequencing runs was >99.7%. Phylogenetic analysis showed that the consensus sequences achieved with Flongle delivered accurate genotyping. We conclude that the new Flongle-based assay with its fast turnover time, low cost investment, and low cost per sample represents an accurate, reproducible, and cost-effective platform for enterovirus identification and genotyping