Read Mapping Research Articles

Prediction of tumor-specific splicing from somatic mutations as a source of neoantigen candidates.

Neoantigens are promising targets for cancer immunotherapies and might arise from alternative splicing. However, detecting tumor-specific splicing is challenging because many non-canonical splice junctions identified in tumors also appear in healthy tissues. To increase tumor-specificity, we focused on splicing caused by somatic mutations as a source for neoantigen candidates in individual patients. We developed the tool splice2neo with multiple functionalities to integrate predicted splice effects from somatic mutations with splice junctions detected in tumor RNA-seq and to annotate the resulting transcript and peptide sequences. Additionally, we provide the tool EasyQuant for targeted RNA-seq read mapping to candidate splice junctions. Using a stringent detection rule, we predicted 1.7 splice junctions per patient as splice targets with a false discovery rate below 5% in a melanoma cohort. We confirmed tumor-specificity using independent, healthy tissue samples. Furthermore, using tumor-derived RNA, we confirmed individual exon-skipping events experimentally. Most target splice junctions encoded neoepitope candidates with predicted major histocompatibility complex (MHC)-I or MHC-II binding. Compared to neoepitope candidates from non-synonymous point mutations, the splicing-derived MHC-I neoepitope candidates had lower self-similarity to corresponding wild-type peptides. In conclusion, we demonstrate that identifying mutation-derived, tumor-specific splice junctions can lead to additional neoantigen candidates to expand the target repertoire for cancer immunotherapies. The R package splice2neo and the python package EasyQuant are available at https://github.com/TRON-Bioinformatics/splice2neo and https://github.com/TRON-Bioinformatics/easyquant, respectively.

An imputed ancestral reference genome for the Mycobacterium tuberculosis complex better captures structural genomic diversity for reference-based alignment workflows.

Reference-based alignment of short-reads is a widely used technique in genomic analysis of the Mycobacterium tuberculosis complex (MTBC) and the choice of reference sequence impacts the interpretation of analyses. The most widely used reference genomes include the ATCC type strain (H37Rv) and the putative MTBC ancestral sequence of Comas et al. both of which are based on a lineage 4 sequence. As such, these reference sequences do not capture all of the structural variation known to be present in the ancestor of the MTBC. To better represent the base of the MTBC, we generated an imputed ancestral genomic sequence, termed MTBC0 from reference-free alignments of closed MTBC genomes. When used as a reference sequence in alignment workflows, MTBC0 mapped more short sequencing reads and called more pairwise SNPs relative to the Comas et al. sequence while exhibiting minimal impact on the overall phylogeny of MTBC. The results also show that MTBC0 provides greater fidelity in capturing genomic variation and allows for the inclusion of regions absent from H37Rv in standard MTBC workflows without additional steps. The use of MTBC0 as an ancestral reference sequence in standard workflows modestly improved read mapping, SNP calling and intuitively facilitates the study of structural variation and evolution in MTBC.

Study and career counsellors: the hub of Swedish adult education

ABSTRACT This article explores how the Swedish policy of municipal adult education (MAE) is interpreted, translated, and enacted in study and career counselling. The data consists of semi-structured interviews with adult education leaders and study and career counsellors. Swedish MAE is characterised by extensive marketisation, with many different providers, which makes the education system complex. The findings show that a study and career counsellor is characterised as a key person for both the students and the organisation, with a role as a marketer and ‘map reader’ for applicants, in addition to responsibilities in admissions, the validation and mapping of students’ knowledge, and quality work. The counsellor becomes a hub in a system that applicants and students find difficult to navigate and fulfils a vital function in the marketing of adult education and quality work. Thus, this complex system requires more resources for counselling to function to the benefit of the individual student. However, there is no clear regulation for how counselling should be organised, which means that counselling is organised in different ways in different municipalities with different providers. This in turn means that the availability of adequate counselling may depend on the municipality in which you live.

Hot alkaline lysis gDNA extraction from formalin-fixed archival tissues.

Formalin fixation of natural history specimens and histopathological material has historically been viewed as an impediment to successful genomic analysis. However, the development of extraction methods specifically tailored to contend with heavily crosslinked archival tissues, re-contextualises millions of previously overlooked specimens as viable molecular assets. Here, we present an easy-to-follow protocol for screening archival wet specimens for molecular viability and subsequent genomic DNA extraction suitable for sequencing. The protocol begins with non-destructive assessment of specimen degradation and preservation media conditions to allow both museum curators and researchers to select specimens most likely to yield an acceptable proportion (20-60%) of mappable endogenous DNA during short-read DNA sequencing. The extraction protocol uses hot alkaline lysis in buffer (0.1M NaOH, 1% SDS, pH 13) to simultaneously lyse and de-crosslink the tissue. To maximise DNA recovery, phenol:chloroform extraction is coupled with a small-fragment optimised SPRI bead clean up. Applied to well-preserved archival tissues, the protocol can yield 1-2 μg DNA per 50 mg of tissue with mean fragment sizes typically ranging from 50-150 bp, which is suitable to recover genomic DNA sufficient to reconstruct complete mitochondrial genomes and achieve up to 25X nuclear genome coverage. We provide guidance for read mapping to a reference genome and discuss the limitations of relying on small fragments for SNP genotyping and de novo genome assembly. This protocol opens the door to broader-scale genetic and phylogenetic analysis of historical specimens, contributing to a deeper understanding of evolutionary trends and adaptation in response to changing environments.

Tour of a Map Reader’s Brain, Part 5: Patterns, Symmetries, Sequences, and Hierarchies

Tour of a Map Reader’s Brain, Part 5: Patterns, Symmetries, Sequences, and Hierarchies

Level of pupils´ map skills in Slovakia

The aim of geographical education is to enhance the level of geographical literacy among pupils, enabling them to acquire, comprehend, analyse, and interpret geographic information so they are able to apply it effectively. One approach to improving this literacy level involves utilizing map skills, through which pupils can read, analyse, interpret map content, and even create maps. This research aimed to assess the level of selected map skills among Slovak pupils at the end of lower secondary education (ISCED 2). A self-constructed research tool was used as the research instrument. Analysis of the acquired data revealed an average level of map skills among the pupils. The highest proficiency was observed in tasks focused on utilizing skills associated with map reading. Conversely, the lowest proficiency was found in tasks requiring the application of skills related to map analysis. The data collected suggest that although pupils at the end of lower secondary education possess skills enabling them to obtain necessary information from a map, they encounter challenges in analysing and subsequently interpreting this information.

DTDHM: detection of tandem duplications based on hybrid methods using next-generation sequencing data.

Tandem duplication (TD) is a common and important type of structural variation in the human genome. TDs have been shown to play an essential role in many diseases, including cancer. However, it is difficult to accurately detect TDs due to the uneven distribution of reads and the inherent complexity of next-generation sequencing (NGS) data. This article proposes a method called DTDHM (detection of tandem duplications based on hybrid methods), which utilizes NGS data to detect TDs in a single sample. DTDHM builds a pipeline that integrates read depth (RD), split read (SR), and paired-end mapping (PEM) signals. To solve the problem of uneven distribution of normal and abnormal samples, DTDHM uses the K-nearest neighbor (KNN) algorithm for multi-feature classification prediction. Then, the qualified split reads and discordant reads are extracted and analyzed to achieve accurate localization of variation sites. This article compares DTDHM with three other methods on 450 simulated datasets and five real datasets. In 450 simulated data samples, DTDHM consistently maintained the highest F1-score. The average F1-score of DTDHM, SVIM, TARDIS, and TIDDIT were 80.0%, 56.2%, 43.4%, and 67.1%, respectively. The F1-score of DTDHM had a small variation range and its detection effect was the most stable and 1.2 times that of the suboptimal method. Most of the boundary biases of DTDHM fluctuated around 20 bp, and its boundary deviation detection ability was better than TARDIS and TIDDIT. In real data experiments, five real sequencing samples (NA19238, NA19239, NA19240, HG00266, and NA12891) were used to test DTDHM. The results showed that DTDHM had the highest overlap density score (ODS) and F1-score of the four methods. Compared with the other three methods, DTDHM achieved excellent results in terms of sensitivity, precision, F1-score, and boundary bias. These results indicate that DTDHM can be used as a reliable tool for detecting TDs from NGS data, especially in the case of low coverage depth and tumor purity samples.

Comparative Methods for Demystifying Spatial Transcriptomics.

Spatial Transcriptomics (ST), coined as the term for parallel RNA-Seq on cell populations ordered spatially on a histological tissue section, has recently become increasingly popular, especially in experiments where microfluidics-based single-cell sequencing fails, such as assays on neurons. ST platforms, like the 10x Visium technology investigated herein, therefore produce in a single experiment simultaneously thousands of RNA readouts, captured by an array of micrometer scale spots under the histological section. Therefore, a central challenge of analyzing ST experiments consists of analyzing the gene expression morphology of all spots to delineate clusters of similar cell mixtures, which are then compared to each other to identify up- or down-regulated marker genes. Moreover, another level of complexity in ST experiments, compared to traditional RNA-Seq, is imposed by staining the tissue section with protein markers of cells or cell components to identify spots providing relevant information afterward. The corresponding microscopy images need to be analyzed in addition to the RNA-Seq read mappings on the reference genome and transcriptome sequences. Focusing on the software suite provided by the Visium platform manufacturer, we break down the ST analysis pipeline into its four essential steps-the image analysis, the read alignment, the gene quantification, and the spot clustering-and compare results obtained when using reads from different subsets of spots and/or when employing alternative genome or transcriptome references. Our comparative analyses demonstrate the impact of spot selection and the choice of genome/transcriptome references on the analysis results when employing the manufacturer's pipeline.

MiniDBG: A Novel and Minimal De Bruijn Graph for Read Mapping.

The De Bruijn graph (DBG) has been widely used in the algorithms for indexing or organizing read and reference sequences in bioinformatics. However, a DBG model that can locate each node, edge and path on sequence has not been proposed so far. Recently, DBG has been used for representing reference sequences in read mapping tasks. In this process, it is not a one-to-one correspondence between the paths of DBG and the substrings of reference sequence. This results in the false path on DBG, which means no substrings of reference producing the path. Moreover, if a candidate path of a read is true, we need to locate it and verify the candidate on sequence. To solve these problems, we proposed a DBG model, called MiniDBG, which stores the position lists of a minimal set of edges. With the position lists, MiniDBG can locate any node, edge and path efficiently. We also proposed algorithms for generating MiniDBG based on an original DBG and algorithms for locating edges or paths on sequence. We designed and ran experiments on real datasets for comparing them with BWT-based and position list-based methods. The experimental results show that MiniDBG can locate the edges and paths efficiently with lower memory costs.

Challenges for teachers in integrating ICT into geography teaching and promoting map reading skills in secondary schools in Namibia

Challenges for teachers in integrating ICT into geography teaching and promoting map reading skills in secondary schools in Namibia

Developing Topographic Map Study Skills in Advanced-Level Geography Students: A Teacher’s Experiences

Map work is a core component of the geography curriculum, but it presents unique challenges for both students and teachers. Students must have a high level of cognitive and conceptual development to read and interpret a map. Teachers, too, often find it difficult to instill the necessary skills for graphic literacy. The unique challenges associated with topographic map study skills are highly relevant to the choice of this subject. The primary goal of this study was to help advanced-level geography students improve their topographic map study skills. For this study, the action research method was used as a research method. Following the pre-test, four students with lower grades were chosen as a sample. Thematic analysis is performed on the data. This research found that most of the students in year 12 felt that they had some issues with incorrect topography identification based on map observation, incorrect recognition of standard symbols from map observation, incorrectly calculating the area of a map, difficulties comprehending the relationship between physical and cultural landscapes, and difficulties with reading and analysing a map. The research then revealed that students require fundamental knowledge of drawing, reading, and interpreting maps. It is also critical to teach map work in as practical a manner as possible. Teachers can use models as teaching aids and the group discussion method as a teaching method to help students develop these skills. Teachers’ peer intervention, individual attention, and discussion with friends all help to increase knowledge of basic topographic map concepts. It contends that the sophistication and complexity of the skills required to interpret topographical maps should not be underestimated. Teachers must be made aware of how students acquire map reading skills and the difficulties they face during this process so that teaching programmes that correspond to students’ levels of cognitive development can be developed. The study’s findings and recommendations have implications for geography teachers, textbook authors, and educational authorities, particularly those involved in curriculum and syllabus development.

A tale of caution: How endogenous viral elements affect virus discovery in transcriptomic data.

Large-scale metagenomic and -transcriptomic studies have revolutionized our understanding of viral diversity and abundance. In contrast, endogenous viral elements (EVEs), remnants of viral sequences integrated into host genomes, have received limited attention in the context of virus discovery, especially in RNA-Seq data. EVEs resemble their original viruses, a challenge that makes distinguishing between active infections and integrated remnants difficult, affecting virus classification and biases downstream analyses. Here, we systematically assess the effects of EVEs on a prototypical virus discovery pipeline, evaluate their impact on data integrity and classification accuracy, and provide some recommendations for better practices. We examined EVEs and exogenous viral sequences linked to Orthomyxoviridae, a diverse family of negative-sense segmented RNA viruses, in 13 genomic and 538 transcriptomic datasets of Culicinae mosquitoes. Our analysis revealed a substantial number of viral sequences in transcriptomic datasets. However, a significant portion appeared not to be exogenous viruses but transcripts derived from EVEs. Distinguishing between transcribed EVEs and exogenous virus sequences was especially difficult in samples with low viral abundance. For example, three transcribed EVEs showed full-length segments, devoid of frameshift and nonsense mutations, exhibiting sufficient mean read depths that qualify them as exogenous virus hits. Mapping reads on a host genome containing EVEs before assembly somewhat alleviated the EVE burden, but it led to a drastic reduction of viral hits and reduced quality of assemblies, especially in regions of the viral genome relatively similar to EVEs. Our study highlights that our knowledge of the genetic diversity of viruses can be altered by the underestimated presence of EVEs in transcriptomic datasets, leading to false positives and altered or missing sequence information. Thus, recognizing and addressing the influence of EVEs in virus discovery pipelines will be key in enhancing our ability to capture the full spectrum of viral diversity.

The Effectiveness of Articulate Storyline 3 Application-Based Interactive Learning Media in Social Studies Learning for Elementary School

The research problem is students' low reading skills and social studies learning outcomes. This research aims to apply interactive media Articulate Storyline 3 to improve students' general map reading skills and learning outcomes in elementary school subjects in Makassar City. This study uses the ASSURE Research and Development (RD) research method (Analyze Learner Characteristics, State Objective Select Media, Utilize Media, Required learner participation Evaluate and revise) to develop learning media that can improve reading skills and learning outcomes for 28 elementary school students in Makassar city. The results showed that interactive learning media was declared effective because two indicators were achieved: the observations of student activities included in the very active category and the observations of teacher activities included in the very active category. Implications in learning can improve skills and student learning outcomes.

Impact of Heuristic Approach on Students’ Academic Achievement and Retention in Map Reading and Interpretation Among Secondary Schools, Municipal Zones, Kano-Nigeria

This study explores the impact of employing a heuristic approach on students’ academic achievement and retention in geography education. The sample consisted of 235 SS II students enrolled in public secondary schools in the Municipal Education Zone, Kano, Nigeria. The study utilized a quasi-experimental design, with two schools assigned to the experimental group (heuristic approach) and two schools assigned to the control group (lecture method). The Geography Achievement Test (GAT) was used as the data collection instrument. ANCOVA was used to test the hypothesis using SPSS and the findings revealed a significant difference in the mean academic achievements between students taught geography concepts using the heuristic approach and those taught using the lecture method. The experimental group exhibited higher academic achievement compared to the control group, indicating that the use of the heuristic approach enhances students’ understanding and performance in geography. Also, there was a significant difference in the mean retention scores of male and female students when taught geography using the heuristic approach. Female students achieved higher retention scores than their male counterparts, suggesting that the heuristic approach is particularly effective in facilitating knowledge retention for female students in geography education. Based on the findings, it is recommended that educators and curriculum developers incorporate the heuristic approach into teaching methods for geography concepts. Training programs and workshops can be conducted to familiarize teachers with effective strategies for implementing the heuristic approach in their lessons.

MCPtaggR: R package for accurate genotype calling in reduced representation sequencing data by eliminating error-prone markers based on genome comparison.

Reduced representation sequencing (RRS) offers cost-effective, high-throughput genotyping platforms such as genotyping-by-sequencing (GBS). RRS reads are typically mapped onto a reference genome. However, mapping reads harbouring mismatches against the reference can potentially result in mismapping and biased mapping, leading to the detection of error-prone markers that provide incorrect genotype information. We established a genotype-calling pipeline named mappable collinear polymorphic tag genotyping (MCPtagg) to achieve accurate genotyping by eliminating error-prone markers. MCPtagg was designed for the RRS-based genotyping of a population derived from a biparental cross. The MCPtagg pipeline filters out error-prone markers prior to genotype calling based on marker collinearity information obtained by comparing the genome sequences of the parents of a population to be genotyped. A performance evaluation on real GBS data from a rice F2 population confirmed its effectiveness. Furthermore, our performance test using a genome assembly that was obtained by genome sequence polishing on an available genome assembly suggests that our pipeline performs well with converted genomes, rather than necessitating de novo assembly. This demonstrates its flexibility and scalability. The R package, MCPtaggR, was developed to provide functions for the pipeline and is available at https://github.com/tomoyukif/MCPtaggR.

Churros: a Docker-based pipeline for large-scale epigenomic analysis.

The epigenome, which reflects the modifications on chromatin or DNA sequences, provides crucial insight into gene expression regulation and cellular activity. With the continuous accumulation of epigenomic datasets such as chromatin immunoprecipitation followed by sequencing (ChIP-seq) data, there is a great demand for a streamlined pipeline to consistently process them, especially for large-dataset comparisons involving hundreds of samples. Here, we present Churros, an end-to-end epigenomic analysis pipeline that is environmentally independent and optimized for handling large-scale data. We successfully demonstrated the effectiveness of Churros by analyzing large-scale ChIP-seq datasets with the hg38 or Telomere-to-Telomere (T2T) human reference genome. We found that applying T2T to the typical analysis workflow has important impacts on read mapping, quality checks, and peak calling. We also introduced a useful feature to study context-specific epigenomic landscapes. Churros will contribute a comprehensive and unified resource for analyzing large-scale epigenomic data.

Genome-resolved metatranscriptomics reveals conserved root colonization determinants in a synthetic microbiota

The identification of processes activated by specific microbes during microbiota colonization of plant roots has been hampered by technical constraints in metatranscriptomics. These include lack of reference genomes, high representation of host or microbial rRNA sequences in datasets, or difficulty to experimentally validate gene functions. Here, we recolonized germ-free Arabidopsis thaliana with a synthetic, yet representative root microbiota comprising 106 genome-sequenced bacterial and fungal isolates. We used multi-kingdom rRNA depletion, deep RNA-sequencing and read mapping against reference microbial genomes to analyse the in planta metatranscriptome of abundant colonizers. We identified over 3,000 microbial genes that were differentially regulated at the soil-root interface. Translation and energy production processes were consistently activated in planta, and their induction correlated with bacterial strains’ abundance in roots. Finally, we used targeted mutagenesis to show that several genes consistently induced by multiple bacteria are required for root colonization in one of the abundant bacterial strains (a genetically tractable Rhodanobacter). Our results indicate that microbiota members activate strain-specific processes but also common gene sets to colonize plant roots.

Crafting cartographic competence: A methodological approach for nurturing future geography teachers

Relevance. The reform of geography education poses new challenges for pre-service geography teacher training programs, particularly in developing cartographic competencies critical for effective teaching.Purpose. The purpose of the study is to examine methods for integrating pedagogical approaches, multimedia tools, and geographic information systems (GIS) to foster cartographic skills in future geography teachers.Methodology. A multimodal approach was implemented, integrating hands-on map reading, analysis of cartographic symbology, engagement with modern technologies like GIS, and field-based activities.Results. The multimodal approach proved effective in building requisite cartographic skills in pre-service teachers. Continuous monitoring and skills upgrading were key factors in preparing teachers capable of translating curricula goals into practical classroom applications. Specific results include improved map interpretation and spatial analysis abilities, proficiency in utilizing GIS software for data visualization, enhanced understanding of cartographic design principles, and the ability to develop engaging map-based learning activities.Conclusions. Developing robust cartographic competencies in pre-service programs enables future geography teachers to leverage maps, spatial representations, and geospatial technologies as powerful pedagogical tools. The immersive, technology-driven methods reflecting contemporary best practices produced educators proficient in cartographic instruction.

Penerapan Alat Bantu Penangkapan Ikan Pada Kelompok Nelayan Pole and Line di Desa Murante Kecamatan Suli Kabupaten Luwu

The objectives of this activity are: 1) To increase catch results and reduce operational costs through the implementation of fishing technology tools. 2) To enhance the capabilities of Pole and Line fishermen in using fishing tools such as GPS, Fish Finder, and the ability to independently read fishing catch maps. The method used in the implementation of Science and Technology for Society (IbM) activities is Participatory Rural Appraisal (PRA), which involves the community in the activities. The implementation of this activity includes counseling, training, demonstrations, and evaluations to assess the program's effectiveness, ensuring efficient socialization of the program. The implementation method involves applying and utilizing fishing tools (GPS, Fish Finder) for Pole and Line fishermen to determine fishing areas and capture fish according to the information on the fishing catch map issued by the Ministry of Marine Affairs and Fisheries of Indonesia. The results of this activity include the dissemination of information on the use of fishing technology tools, training in reading maps of potential fishing zones, and assistance in using the fish finder through counseling, direct practice, and interactive discussions to uncover issues and solutions that can be addressed by partners. In conclusion, the outcomes of the activity are: 1) Increased catch results and reduced operational costs through the implementation of fishing technology tools. 2) The desire and ability of Pole and Line fishermen to use fishing tools such as GPS, Fish Finder, and independently read fishing catch maps, subsequently inputting this information into the fishing tools

Dancing the Nanopore limbo – Nanopore metagenomics from small DNA quantities for bacterial genome reconstruction

BackgroundWhile genome-resolved metagenomics has revolutionized our understanding of microbial and genetic diversity in environmental samples, assemblies of short-reads often result in incomplete and/or highly fragmented metagenome-assembled genomes (MAGs), hampering in-depth genomics. Although Nanopore sequencing has increasingly been used in microbial metagenomics as long reads greatly improve the assembly quality of MAGs, the recommended DNA quantity usually exceeds the recoverable amount of DNA of environmental samples. Here, we evaluated lower-than-recommended DNA quantities for Nanopore library preparation by determining sequencing quality, community composition, assembly quality and recovery of MAGs.ResultsWe generated 27 Nanopore metagenomes using the commercially available ZYMO mock community and varied the amount of input DNA from 1000 ng (the recommended minimum) down to 1 ng in eight steps. The quality of the generated reads remained stable across all input levels. The read mapping accuracy, which reflects how well the reads match a known reference genome, was consistently high across all libraries. The relative abundance of the species in the metagenomes was stable down to input levels of 50 ng. High-quality MAGs (> 95% completeness, ≤ 5% contamination) could be recovered from metagenomes down to 35 ng of input material. When combined with publicly available Illumina reads for the mock community, Nanopore reads from input quantities as low as 1 ng improved the quality of hybrid assemblies.ConclusionOur results show that the recommended DNA amount for Nanopore library preparation can be substantially reduced without any adverse effects to genome recovery and still bolster hybrid assemblies when combined with short-read data. We posit that the results presented herein will enable studies to improve genome recovery from low-biomass environments, enhancing microbiome understanding.