Classificier of reads from metagenomic sequencing experiments.

•  Kawulok, J., Deorowicz, S., CoMeta: Classification of Metagenomes Using k-mers, PLOS ONE, 2015; 10(4):1–23,

CoMSA

Compressor of multiple sequence alignments of proteins.

•  Deorowicz, S., Walczyszyn, J., Debudaj-Grabysz, A., CoMSA: compression of protein multiple sequence alignment files, Bioinformatics, 2019; 35(2):22–234,

DSRC

Compressor of sequencing reads.

•  Roguski, L., Deorowicz, S., DSRC 2: Industry-oriented compression of FASTQ files, Bioinformatics, 2014; 30(15):2213–2215,
•  Deorowicz, S., Grabowski, Sz., Compression of DNA sequences in FASTQ format, Bioinformatics, 2011; 27(6):860–862,

FAMSA

Multiple sequence alignment designed for huge families of proteins (even containing hundreds of thousands of sequences).

•  Deorowicz, S., Debudaj-Grabysz, A., Gudys, A., FAMSA: Fast and accurate multiple sequence alignment of huge protein families, Scientific Reports, 2016; 6(33964):

FaStore

Compressor of FASTQ files.

•  Roguski, L., Ochoa, I., Hernaez, M., Deorowicz, S., FaStore - a space-saving solution for raw sequencing data, Bioinformatics, 2018; 34(16):2748–2756,

FQSqueezer

Experimental high-end compressor of FASTQ files.

•  Deorowicz, S., FQSqueezer: k-mer-based compression of sequencing data, Scientific Reports, 2020; 10(578):

GDC

Compressor of collections of genome sequences.

•  Deorowicz, S., Danek, A., Niemiec, M., GDC 2: Compression of large collections of genomes, Scientific Reports, 2015; 5(11565):1–12,
•  Deorowicz, S., Grabowski, Sz., Robust relative compression of genomes with random access, Bioinformatics, 2011; 27(21):2979–2986,

GTC

Genotype databases compressor with support for fast queries.

•  Danek, A., Deorowicz, S., GTC: how to maintain huge genotype collections in a compressed form, Bioinformatics, 2018; 34(11):1834–1840,

GTShark

Genotypes compressor.

•  Deorowicz, S., Danek, A., GTShark: Genotype compression in large projects, Bioinformatics, 2019; 35(22):4791–4793,

KMC

Memory frugal k-mer counter.

•  Kokot, M., Długosz, M., Deorowicz, S., KMC 3: counting and manipulating k -mer statistics, Bioinformatics, 2017; 33(17):2759–2761,
•  Deorowicz, S., Kokot, M., Grabowski, Sz., Debudaj-Grabysz, A., KMC 2: Fast and resource-frugal k-mer counting, Bioinformatics, 2015; 31(10):1569–1576,
•  Deorowicz, S., Debudaj-Grabysz, A., Grabowski, Sz., Disk-based k-mer counting on a PC, BMC Bioinformatics, 2013; 14():Article no. 160,

Kmer-db

Tool for estimation of evolutionary distances in a collection of genomes.

•  Deorowicz, S., Gudys, A., Dlugosz, M., Kokot, M., Danek, A., Kmer-db: instant evolutionary distance estimation, Bioinformatics, 2019; 35(1):133–136,

MuGI

Index allowing queries for a collection of multiple genome sequences.

•  Danek, A., Deorowicz, S., Grabowski, Sz., Indexes of Large Genome Collections on a PC, PLOS ONE, 2014; 9(10):e109384,

ORCOM

Experimental compressor of sequencing reads.

•  Grabowski, Sz., Deorowicz, S., Roguski, L., Disk-based compression of data from genome sequencing, Bioinformatics, 2014; 31(9):1389–1395,

PgSA

Index allowing queries for a collection of sequencing reads.

•  Kowalski, T., Grabowski, Sz., Deorowicz, S., Indexing arbitrary-length k-mers in sequencing reads, PLOS ONE, 2015; 10(7):1–16,

QuickProbs

Multiple sequence alignment designed especially for GPU.

•  Gudys, A., Deorowicz, S., QuickProbs 2: towards rapid construction of high-quality alignments of large protein families, Scientific Reports, 2017; 7(41553):
•  Gudys, A., Deorowicz, S., QuickProbs – A Fast Multiple Sequence Alignment Algorithm Designed for Graphics Processors, PLOS ONE, 2014; 9(2):e88901,

RECKONER

Read error corrector.

•  Maciej Długosz, M., Deorowicz, S., RECKONER: read error corrector based on KMC, Bioinformatics, 2017; 33(7):1086–1089,

TGC

Compressor of collections of genomes given in Variant Call Format (VCF) files.

•  Deorowicz, S., Danek, A., Grabowski, Sz., Genome compression: a novel approach for large collections, Bioinformatics, 2013; 29(20):2572–2578,

VCFShark

Compressor of VCF files.

•  Deorowicz, S., Danek, A., GTShark: Genotype compression in large projects, biorxiv.org, 2020; ():

Whisper

Experimental mapper of whole genome sequencing data.

•  Deorowicz, S., Gudys, A., Whisper 2: indel-sensitive short read mapping, bioRxiv.org, 2019; :
•  Deorowicz, S., Debudaj-Grabysz, A., Gudys, A., Grabowski, Sz., Whisper: read sorting allows robust robust mapping of DNA sequencing data, Bioinformatics, 2019; 35(12):2043–2050,
•  Deorowicz, S., Debudaj-Grabysz, A., Gudys, A., Grabowski, Sz., Robust mapping of whole genome sequencing data, Poster at The Biology of Genomes Conference, 2017;

1. MISA: MISA (MIcroSAtellite) is a web-based tool that can identify SSRs in DNA sequences. It can be used to analyze nucleotide sequences from various organisms and can identify perfect, compound, and imperfect SSRs.

2. SSR Locator: SSR Locator is a web-based tool that identifies SSRs in both DNA and RNA sequences. It can identify perfect, compound, and imperfect SSRs, and can also filter out low complexity regions.

3. SciRoKo: SciRoKo is a software tool that can identify SSRs in DNA sequences. It can be used to analyze genomic and transcriptomic sequences from various organisms and can identify perfect, compound, and imperfect SSRs.

4. Primer3: Primer3 is a web-based tool that designs PCR primers for SSRs. It can design primers for perfect and imperfect SSRs, and can be used to design primers for SSRs in various organisms.

5. QDD: QDD (Quick Detection of Duplication) is a software tool that can identify SSRs in DNA sequences and can also identify duplicate loci. It can be used to analyze genomic and transcriptomic sequences from various organisms.

These are just a few examples of the many bioinformatics tools available for exploring SSRs. Depending on your specific needs and research questions, you may find that other tools are more appropriate for your analysis.

What is Bacterial Comparative Genomics?

Comparative genomics involves the systematic comparison of genomes across different bacterial species or strains. This approach allows scientists to:

• Identify conserved and unique genes.

• Explore genetic determinants of pathogenicity.

• Understand bacterial evolution and phylogenetics.

• Investigate horizontal gene transfer and its role in antibiotic resistance.

Bioinformatics is central to these analyses, enabling the processing and interpretation of large-scale genomic data.

Key Steps in Bacterial Comparative Genomics

1. Genome Sequencing and Assembly: The process begins with obtaining high-quality bacterial genome sequences. Advances in next-generation sequencing (NGS) technologies have made it faster and more affordable to sequence bacterial genomes. Tools such as SPAdes and Velvet are commonly used for genome assembly.

2. Genome Annotation: Annotating a genome involves identifying genes, regulatory elements, and other genomic features. Automated tools like Prokka and RAST provide functional annotations, allowing researchers to predict the roles of genes and proteins.

3. Genome Alignment: Aligning genomes is crucial for identifying conserved regions, single-nucleotide polymorphisms (SNPs), and structural variations. Tools like Mauve and progressiveMauve are commonly employed for whole-genome alignments.

4. Comparative Analyses:

   • Core and Pan-genome Analysis: The core genome consists of genes shared across all strains of a species, while the pan-genome includes all genes found in any strain. Software like Roary and BPGA can perform core and pan-genome analyses.

   • Phylogenetic Analysis: Comparative genomics often involves reconstructing evolutionary relationships. Tools such as MEGA and IQ-TREE facilitate phylogenetic tree construction based on genomic data.

   • Functional Enrichment Analysis: To understand the biological significance of unique or shared genes, functional enrichment analysis using databases like GO (Gene Ontology) and KEGG is essential.

Here are some additional bioinformatics tools that can aid bacterial comparative genomics:

• OrthoFinder: For accurate ortholog identification across multiple genomes.

• PanOCT: Specifically designed for pan-genome clustering and annotation.

• FASTANI: A tool for calculating Average Nucleotide Identity (ANI) for microbial genome comparisons.

• CIRCOS: For visually comparing genomic data through circular genome plots.

• Galaxy Platform: A user-friendly web-based platform offering numerous genomic analysis tools.

• BLAST: Essential for sequence alignment and similarity searches.

• PhyloSift: Focused on phylogenetic analysis of microbial genomes using marker genes.

These tools, in combination with the methods discussed, provide a robust framework for conducting comprehensive comparative genomic studies.

Applications of Bacterial Comparative Genomics

1. Understanding Pathogenicity: Comparative genomics helps identify virulence factors that distinguish pathogenic strains from non-pathogenic relatives. For instance, comparing genomes of Escherichia coli strains has revealed key genetic determinants of pathogenicity in enterohemorrhagic strains.

2. Antibiotic Resistance Research: The spread of antibiotic resistance genes through horizontal gene transfer is a major global concern. Comparative analyses can trace the origins and dissemination of resistance genes, aiding in the development of countermeasures.

3. Microbial Ecology and Evolution: By studying genomic variations, researchers can understand how bacteria adapt to different environments. This is particularly relevant for extremophiles and symbiotic bacteria.

4. Vaccine Development: Identifying conserved antigens across pathogenic strains is critical for vaccine design. Comparative genomics has been instrumental in developing vaccines against pathogens like Neisseria meningitidis.

5. Biotechnology Applications: Comparative studies can uncover unique metabolic pathways in bacteria, paving the way for applications in bioremediation, synthetic biology, and industrial microbiology.

Challenges in Bacterial Comparative Genomics

While the field has made significant strides, several challenges remain:

• Data Overload: The rapid growth of sequencing data requires robust computational infrastructure and efficient algorithms.

• Genome Plasticity: High rates of horizontal gene transfer and genome rearrangements in bacteria complicate comparative analyses.

• Annotation Accuracy: Automated annotation tools are not infallible, and manual curation is often needed for high-confidence results.

• Interpreting Non-Coding Regions: Understanding the functional significance of non-coding genomic regions remains a challenge.

Future Directions

The integration of bacterial comparative genomics with other ‘omics’ approaches—such as transcriptomics, proteomics, and metabolomics—promises a more comprehensive understanding of bacterial biology. Additionally, advancements in machine learning and artificial intelligence are likely to further enhance bioinformatics analyses, enabling the prediction of complex phenotypes from genomic data.

Conclusion

Bacterial comparative genomics, driven by bioinformatics, continues to unravel the complexities of bacterial life. From combating antibiotic resistance to uncovering the secrets of microbial evolution, this interdisciplinary field holds immense potential for addressing pressing challenges in microbiology and beyond. As technology advances, so too will our ability to harness the power of comparative genomics for scientific and societal benefit.

• BigDataScript – A cross-system scripting language for working with big data pipelines in computer systems of different sizes and capabilities. [ paper-2014 | web ]
• Bpipe – A small language for defining pipeline stages and linking them together to make pipelines. [ web ]
• Common Workflow Language – a specification for describing analysis workflows and tools that are portable and scalable across a variety of software and hardware environments, from workstations to cluster, cloud, and high performance computing (HPC) environments. [ web ]
• Cromwell – A Workflow Management System geared towards scientific workflows. [ web ]
• Galaxy – a popular open-source, web-based platform for data intensive biomedical research. Has several features, from data analysis to workflow management to visualization tools. [ paper-2018 | web ]
• Nextflow (recommended) – A fluent DSL modelled around the UNIX pipe concept, that simplifies writing parallel and scalable pipelines in a portable manner. [ paper-2018 | web ]
• Ruffus – Computation Pipeline library for python widely used in science and bioinformatics. [ paper-2010 | web ]
• SeqWare – Hadoop Oozie-based workflow system focused on genomics data analysis in cloud environments. [ paper-2010 | web ]
• Snakemake – A workflow management system in Python that aims to reduce the complexity of creating workflows by providing a fast and comfortable execution environment. [ paper-2018 | web ]
• Workflow Descriptor Language – Workflow standard developed by the Broad. [ web ]

However, nanopore sequencing is an emerging and rapidly developing technology. It is not clear what will be most informative. We hope that IONiseR will provide a framework for visualisation of metrics that we haven’t thought of, and welcome feedback at mike.smith@embl.de.

If you’re not interested in the quality assement of the raw or event level data, and want to jump straight to the getting FASTQ format files from fast5 files you can go straight to the final section of this document.

Address of the bookmark: https://www.bioconductor.org/packages/devel/bioc/vignettes/IONiseR/inst/doc/IONiseR.html

ABySS Explorer

Interactive Java application that uses a novel graph-based representation to display a sequence assembly and associated metadata

http://www.bcgsc.ca/platform/bioinfo/software/abyss-explorer

BamView

Genome browser and annotation tool that allows visualization of sequence features, next-generation sequencing (NGS) data and the results of analyses within the context of the sequence, and also its six-frame translation

http://www.sanger.ac.uk/resources/software/artemis/

DNannotator 

Annotation web toolkit for regional genomic sequences

http://bioapp.psych.uic.edu/DNannotator.htm

JVM 

Java Visual Mapping tool for NGS reads

http://www.springer.com/cda/content/document/cda_downloaddocument/9789401792448-c2.pdf?SGWID=0-0-45-1487072-p176815501

LookSeq 

Web-based visualization of sequences derived from multiple sequencing technologies. Low- or high-depth read pileups and easy visualization of putative single nucleotide and structural variation

http://lookseq.sourceforge.net

MagicViewer 

Visualization of short read alignment, identification of genetic variation and association with annotation information of a reference genome

http://bioinformatics.zj.cn/magicviewer/

MapView 

Alignments of huge-scale single-end and pair-end short reads

http://omictools.com/mapview-s1367.html

MultiPipMaker

Computes alignments of similar regions in two DNA sequences. The resulting alignments are summarized with a ‘percent identity plot’ (pip)

http://pipmaker.bx.psu.edu/pipmaker/

PileLineGUI 

Handling genome position files in NGS studies

http://sing.ei.uvigo.es/pileline/pilelinegui.html

SAMtools tview 

Simple and fast text alignment viewer; NGS compatible

http://www.htslib.org/

SEWAL

Uses a locality-sensitive hashing algorithm to enumerate all unique sequences in an entire Illumina sequencing run

http://www.sourceforge.net/projects/sewal

STAR 

A web-based integrated solution to management and visualization of sequencing data

http://wanglab.ucsd.edu/star/browser

SVA 

Software for annotating and visualizing sequenced human genomes

http://www.svaproject.org

Viewer (IGV) 

Visualization of large heterogeneous datasets, providing a smooth and intuitive user experience at all levels of genome resolution

https://www.broadinstitute.org/igv/

ZOOM Lite 

NGS data mapping and visualization software

http://bioinfor.com/zoom/lite/

Take a look at the PRICE software from the DeRisi lab. Its meant to do something very similar. http://derisilab.ucsf.edu/index.php?page=software

You could also look at SSPACE (http://www.baseclear.com/landingpages/basetools-a-wide-range-of-bioinformatics-solutions/sspacev12/), ATLAS tools (http://www.hgsc.bcm.tmc.edu/content/bcm-hgsc-software), and SCARPA (http://compbio.cs.toronto.edu/hapsembler/scarpa.html).

See the PAGIT protocol: http://www.sanger.ac.uk/resources/software/pagit/

In particular, take a look at the IMAGE tool: http://genomebiology.com/2010/11/4/R41

Also SOAPdenovo has ha function for scaffolding. Not sure about ABYSS

Here there is a useful explanation of several tools.

https://bioinformaticsonline.com/search?q=scaffolding&entity_type=object&entity_subtype=bookmarks&offset=0&search_type=entities

I could be wrong, but the above answers to your hypothetical scenario appear to miss the point that you aren't interested in assembling the full genome, just the 100 kb part you're interested in. I suggest the following algorithm:

1. Start with the initial assembly C0 of the contigs you have identified as overlapping your region of interest, and the set S of reads those contigs contain. Let C = C0.

2. Repeat:
a. Identify paired-end reads (not in C) for which one or both ends align within, or extending, contigs in C.
b. Identify unpaired reads that align extending these new paired-end reads.
c. Construct a new assembly C' from C and the new reads identified in (a) and (b).
d. Trim C' so it does not extend more than 100 kb to either end of C0. Set C = C'.
e. Let S' denote the reads that contribute to C'. If S' does not contain any reads not present in S, stop. Otherwise, Set S = S'.

3. If you don't have a complete assembly of the region of interest, generate an STS for each end of each contig, probe a library for clones including these STSes, subclone these clones into a paired-end sequencing vector, and generate paired-end reads for this library; then try steps (1) and (2) again, adding these new sequencing reads to what you had before.

4. If your average sequencing depth for the region of interest exceeds 25 or so without filling all gaps, it is likely that the remaining gaps represent sequences that are not getting cloned in your sequencing vectors. Try different sequencing vectors.

Address of the bookmark: https://www.molinspiration.com/

https://function.princeton.edu/

1 Falcon https://github.com/PacificBiosciences/pb-assembly

2 Canu assembler http://canu.readthedocs.io/en/latest/index.html

3 Miniasm assembler https://github.com/lh3/miniasm

4 PBJelly scaffolding tool https://sourceforge.net/projects/pb-jelly/

5 ARCS scaffolding tool https://github.com/bcgsc/arcs

6 Redundans reduction and scaffolding tool https://github.com/Gabaldonlab/redundans

7 Arrow error correction https://github.com/PacificBiosciences/ GenomicConsensus

8 PILON error correction https://github.com/broadinstitute/pilon/wiki

9 BUSCO single copy gene markers http://busco.ezlab.org/

10 Bandage graph assembly viewer https://rrwick.github.io/Bandage/

11 Gepard dotter http://cube.univie.ac.at/gepard

12 MUMmer aligner and plotter http://mummer.sourceforge.net/