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1. EDGE COVID-19: a web platform to generate submission-ready genomes from SARS-CoV-2 sequencing efforts

   https://doi.org/10.1093/bioinformatics/btac176  

   Lo, Chien-Chi ; Shakya, Migun ; Connor, Ryan ; Davenport, Karen ; Flynn, Mark ; Gutiérrez, Adán Myers y. ; Hu, Bin ; Li, Po-E ; Jackson, Elais Player ; Xu, Yan ; et al ( March 2022 , Bioinformatics)

   Genomics has become an essential technology for surveilling emerging infectious disease outbreaks. A range of technologies and strategies for pathogen genome enrichment and sequencing are being used by laboratories worldwide, together with different and sometimes ad hoc, analytical procedures for generating genome sequences. A fully integrated analytical process for raw sequence to consensus genome determination, suited to outbreaks such as the ongoing COVID-19 pandemic, is critical to provide a solid genomic basis for epidemiological analyses and well-informed decision making. We have developed a web-based platform and integrated bioinformatic workflows that help to provide consistent high-quality analysis of SARS-CoV-2 sequencing data generated with either the Illumina or Oxford Nanopore Technologies (ONT). Using an intuitive web-based interface, this workflow automates data quality control, SARS-CoV-2 reference-based genome variant and consensus calling, lineage determination and provides the ability to submit the consensus sequence and necessary metadata to GenBank, GISAID and INSDC raw data repositories. We tested workflow usability using real world data and validated the accuracy of variant and lineage analysis using several test datasets, and further performed detailed comparisons with results from the COVID-19 Galaxy Project workflow. Our analyses indicate that EC-19 workflows generate high-quality SARS-CoV-2 genomes. Finally, we share a perspective on patterns and impact observed with Illumina versus ONT technologies on workflow congruence and differences.

   https://edge-covid19.edgebioinformatics.org, and https://github.com/LANL-Bioinformatics/EDGE/tree/SARS-CoV2.

   Supplementary data are available at Bioinformatics online.

    

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2. KARGAMobile: Android app for portable, real-time, easily interpretable analysis of antibiotic resistance genes via nanopore sequencing

   https://doi.org/10.3389/fbioe.2022.1016408  

   Barquero, Alexander ; Marini, Simone ; Boucher, Christina ; Ruiz, Jaime ; Prosperi, Mattia ( October 2022 , Frontiers in Bioengineering and Biotechnology)

   Nanopore technology enables portable, real-time sequencing of microbial populations from clinical and ecological samples. An emerging healthcare application for Nanopore includes point-of-care, timely identification of antibiotic resistance genes (ARGs) to help developing targeted treatments of bacterial infections, and monitoring resistant outbreaks in the environment. While several computational tools exist for classifying ARGs from sequencing data, to date (2022) none have been developed for mobile devices. We present here KARGAMobile, a mobile app for portable, real-time, easily interpretable analysis of ARGs from Nanopore sequencing. KARGAMobile is the porting of an existing ARG identification tool named KARGA; it retains the same algorithmic structure, but it is optimized for mobile devices. Specifically, KARGAMobile employs a compressed ARG reference database and different internal data structures to save RAM usage. The KARGAMobile app features a friendly graphical user interface that guides through file browsing, loading, parameter setup, and process execution. More importantly, the output files are post-processed to create visual, printable and shareable reports, aiding users to interpret the ARG findings. The difference in classification performance between KARGAMobile and KARGA is minimal (96.2% vs . 96.9% f-measure on semi-synthetic datasets of 1 million reads with known resistance ground truth). Using real Nanopore experiments, KARGAMobile processes on average 1 GB data every 23–48 min (targeted sequencing - metagenomics), with peak RAM usage below 500MB, independently from input file sizes, and an average temperature of 49°C after 1 h of continuous data processing. KARGAMobile is written in Java and is available at https://github.com/Ruiz-HCI-Lab/KargaMobile under the MIT license. 

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3. NanoSNP: a progressive and haplotype-aware SNP caller on low-coverage nanopore sequencing data

   https://doi.org/10.1093/bioinformatics/btac824  

   Huang, Neng ; Xu, Minghua ; Nie, Fan ; Ni, Peng ; Xiao, Chuan-Le ; Luo, Feng ; Wang, Jianxin ( January 2023 , Bioinformatics)

   Birol, Inanc (Ed.)

   Abstract Motivation Oxford Nanopore sequencing has great potential and advantages in population-scale studies. Due to the cost of sequencing, the depth of whole-genome sequencing for per individual sample must be small. However, the existing single nucleotide polymorphism (SNP) callers are aimed at high-coverage Nanopore sequencing reads. Detecting the SNP variants on low-coverage Nanopore sequencing data is still a challenging problem. Results We developed a novel deep learning-based SNP calling method, NanoSNP, to identify the SNP sites (excluding short indels) based on low-coverage Nanopore sequencing reads. In this method, we design a multi-step, multi-scale and haplotype-aware SNP detection pipeline. First, the pileup model in NanoSNP utilizes the naive pileup feature to predict a subset of SNP sites with a Bi-long short-term memory (LSTM) network. These SNP sites are phased and used to divide the low-coverage Nanopore reads into different haplotypes. Finally, the long-range haplotype feature and short-range pileup feature are extracted from each haplotype. The haplotype model combines two features and predicts the genotype for the candidate site using a Bi-LSTM network. To evaluate the performance of NanoSNP, we compared NanoSNP with Clair, Clair3, Pepper-DeepVariant and NanoCaller on the low-coverage (∼16×) Nanopore sequencing reads. We also performed cross-genome testing on six human genomes HG002–HG007, respectively. Comprehensive experiments demonstrate that NanoSNP outperforms Clair, Pepper-DeepVariant and NanoCaller in identifying SNPs on low-coverage Nanopore sequencing data, including the difficult-to-map regions and major histocompatibility complex regions in the human genome. NanoSNP is comparable to Clair3 when the coverage exceeds 16×. Availability and implementation https://github.com/huangnengCSU/NanoSNP.git. Supplementary information Supplementary data are available at Bioinformatics online. 

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4. lordFAST: sensitive and Fast Alignment Search Tool for LOng noisy Read sequencing Data

   https://doi.org/10.1093/bioinformatics/bty544  

   Haghshenas, Ehsan ; Sahinalp, S. Cenk ; Hach, Faraz ; Berger, ed., Bonnie ( July 2018 , Bioinformatics)

   Recent advances in genomics and precision medicine have been made possible through the application of high throughput sequencing (HTS) to large collections of human genomes. Although HTS technologies have proven their use in cataloging human genome variation, computational analysis of the data they generate is still far from being perfect. The main limitation of Illumina and other popular sequencing technologies is their short read length relative to the lengths of (common) genomic repeats. Newer (single molecule sequencing – SMS) technologies such as Pacific Biosciences and Oxford Nanopore are producing longer reads, making it theoretically possible to overcome the difficulties imposed by repeat regions. Unfortunately, because of their high sequencing error rate, reads generated by these technologies are very difficult to work with and cannot be used in many of the standard downstream analysis pipelines. Note that it is not only difficult to find the correct mapping locations of such reads in a reference genome, but also to establish their correct alignment so as to differentiate sequencing errors from real genomic variants. Furthermore, especially since newer SMS instruments provide higher throughput, mapping and alignment need to be performed much faster than before, maintaining high sensitivity.

   We introduce lordFAST, a novel long-read mapper that is specifically designed to align reads generated by PacBio and potentially other SMS technologies to a reference. lordFAST not only has higher sensitivity than the available alternatives, it is also among the fastest and has a very low memory footprint.

   lordFAST is implemented in C++ and supports multi-threading. The source code of lordFAST is available at https://github.com/vpc-ccg/lordfast.

   Supplementary data are available at Bioinformatics online.

    

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5. Real-time mapping of nanopore raw signals

   https://doi.org/10.1093/bioinformatics/btab264  

   Zhang, Haowen ; Li, Haoran ; Jain, Chirag ; Cheng, Haoyu ; Au, Kin Fai ; Li, Heng ; Aluru, Srinivas ( July 2021 , Bioinformatics)

   Abstract Motivation Oxford Nanopore Technologies sequencing devices support adaptive sequencing, in which undesired reads can be ejected from a pore in real time. This feature allows targeted sequencing aided by computational methods for mapping partial reads, rather than complex library preparation protocols. However, existing mapping methods either require a computationally expensive base-calling procedure before using aligners to map partial reads or work well only on small genomes. Results In this work, we present a new streaming method that can map nanopore raw signals for real-time selective sequencing. Rather than converting read signals to bases, we propose to convert reference genomes to signals and fully operate in the signal space. Our method features a new way to index reference genomes using k-d trees, a novel seed selection strategy and a seed chaining algorithm tailored toward the current signal characteristics. We implemented the method as a tool Sigmap. Then we evaluated it on both simulated and real data and compared it to the state-of-the-art nanopore raw signal mapper Uncalled. Our results show that Sigmap yields comparable performance on mapping yeast simulated raw signals, and better mapping accuracy on mapping yeast real raw signals with a 4.4× speedup. Moreover, our method performed well on mapping raw signals to genomes of size >100 Mbp and correctly mapped 11.49% more real raw signals of green algae, which leads to a significantly higher F1-score (0.9354 versus 0.8660). Availability and implementation Sigmap code is accessible at https://github.com/haowenz/sigmap. Supplementary information Supplementary data are available at Bioinformatics online. 

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