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We finish our discussion on bacterial taxonomy, this time looking at new approaches of naming the multitudes of unnamed uncultured organisms and the controversial renaming of phyla. With guests Professor Phil Hugenholtz, Professor Iain Sutcliffe and Professor Mark Pallen.

Selective bibliography: 
https://github.com/MicroBinfie/MicroBinfie.github.io/blob/45db8eb57d732176449073065dbdacc88a288fe9/assets/Taxonomy_Selective_bibliography.pdf

71 Bacterial taxonomy: Into the unknown, SeqCode, naming uncultured multitudes and renaming of phyla

90 Public health bioinformatics on the cloud with WDL + Terra.bio

We go through bug reports and issues and give insights into how bioinformaticians dig into them. We suggest the underlying problems and possible solutions and also provide tips on how to file better bug reports.

93 Roary Troubleshooting And Issues

We are talking about KRAKEN - the taxonomic classification software and in the hot seat are Dr Jennifer Lu and Natalia Rincon from Johns Hopkins University Center for Computational Biology.

The MicroBinfie podcast welcomed Dr. Jennifer Lu and Natalia Rincon to discuss Kraken, a taxonomic classification software. Developed in 2013-2014, Kraken easily identifies and assigns sequencing reads to a specific species, genus, or general bacteria. Its efficiency in classifying millions or billions of reads puts it ahead of other classification methods such as Melan, Mega Blast, and Chime. The tool is known for its ease of use and accuracy. 

Following the success of Kraken's metagenomic analysis, Florian Breitweiser developed Kraken Unique, which provides more information than the standard Kraken. C Another edition to the Kraken family is Bracken, developed by Jennifer Lu, which estimates abundance, and Nat Rincon contributes to the newest editions, which analyze diversity metrics.

Kraken's exact camera matching technology identifies reads and classifies taxonomy IDs, with two outputs: a long text file for every read and a Kraken report that provides a breakdown of reads for each taxonomy ID. The interpretation of the Kraken report relies on the sample and its taxon. Even if there are few reads available, taxons can still be meaningful. For beginners, Kraken simplifies the classification process by providing pre-built databases.

There was an interesting discussion about the origin of the Kraken name. It is derived from a mythological creature that relied on Jellyfish, a camera counting tool used to build the Kraken databases. Derek Wood developed the original concept of Kraken.

The hosts found a true pathogen in a sample, which was significant for downstream analysis. The number of reads in some samples was very few, and some unclassified reads could also be uninformative or indicate contamination. Being developed for Illumina reads, Kraken's accuracy in classifying Nanopore reads is likely to be affected due to the higher error rate. The Kraken database achieves exact matching of k-mers and fits all genome information into a small space. Tools spawned out of the Kraken world are widely used due to their high accuracy, speed, and simplicity in the classification of taxonomy.

Kraken provides an additional column in the report to count the number of unique k-mers to validate the results. The developers worked closely with others to test new Nanopore chemistries due to the frequent changes in the chemistry that affected the accuracy of the reads.

Kraken databases contain vector sequence information, and vectors are given their taxonomy ID as "synthetic sequences." The software mixes Pearl and C++, with Pearl processing inputs and C++ managing heavy memory stuff by building and compacting sequences and writing bytes. Dr. Jennifer Lu appreciates the simplicity and accuracy of the classification algorithm, and Nat Rincon takes pride in being part of the Kraken community.

103 Release the Kraken

We talk about KRAKEN the taxonomic classification software and the software suite around it and are joined by Jennifer Lu and Natalia Rincon from Johns Hopkins University Center for Computational Biology.

Dr. Jennifer Lu and Natalia Rincon from the Kraken software development team were interviewed on the MicroBinfie podcast. They discussed the various versions of Kraken and the tools developed around it. They began by explaining the original Kraken, which uses an exact camera matching process and a camera size of 31 based on jellyfish. Kraken Unique is an additional version of Kraken that includes an additional column called unique camera counting, which determines how many unique cameras are covered by each read, providing an additional way to verify microbial identification. Kraken two was developed to accommodate larger databases by using a probabilistic data structure and minimizers to map cameras to a shorter sequence size. 

They then talked about how Kraken is useful for microbiome analysis, including detecting pathogens. However, the accuracy of the results depends heavily on the availability of genomic data in the database, which emphasizes bacterial and viral data. For infectious pathogen detection, Kraken one unique is combined with Bracken to approximate the abundance of species present.

The developers emphasized the importance of users being aware of available genomic data in the database because the results can only be as accurate as the data. They also talked about how Kraken is used widely in bioinformatics and can be used for various scenarios beyond metagenomics. For example, they use Kraken to treat a single genome as a metagenome as part of quality control analysis. In cases where there are conflicting taxa in the reads, Kraken results show it, making it useful in determining the presence of contamination in samples.

The Kraken team also talked about how they use Kraken for contamination work to detect contamination in pathogen genomes. They compare all eukaryotic pathogen genomes against bacteria, human genomes, and databases of vertebrates and plants to filter out any contaminants. They have found in some instances where contaminating sequences from hosts such as chicken or cow were present in eukaryotic pathogen genomes.

Moving forward, the Kraken team intends to maintain all Kraken repositories, enhance its accuracy, speed, and usefulness, and develop new scripts and downstream analysis for the Kraken Tools suite. They acknowledge the need to make the database smaller as more genomes become available and are exploring ways of indexing and sketching to achieve this.

In conclusion, Kraken has been an essential software for metagenomic analysis, and it remains a continually improving tool for pathogen detection and classification. The Kraken team advises users to keep in mind the importance of accurate data for effective pathogen detection and classification.

104 The Kraken software suite

Andrew and Lee are at the Global Microbial Identifier conference 13 in Vancouver Canada. On the first day they talked to Dr Finlay Maguire and Dr Emma Griffiths about microbial genomics and Tim Hortons.

111 Global Microbial Identifier with Finlay Maguire and Emma Griffiths

A quick update from Andrew from the Global Microbial Identifier conference in Vancouver, Canada.

Ep8 Global Microbial Identifier in a nutshell

The podcast, set against the picturesque backdrop of Vancouver, delves into the intricate world of bioinformatics, emphasizing the pivotal role of the Global Microbial Identifier (GMI) in facilitating discussions on data sharing, public health, and international collaborations. Organizers Emma and Will, along with other professionals underscore the challenges in data sharing due to varied international laws, privacy concerns, and technical roadblocks. A significant highlight was the discourse on the importance of respecting the data rights of indigenous and marginalized communities, particularly the First Nations, and the ethical considerations surrounding it. Moreover, the collaboration with Lebanon on SARS-CoV-2 research exemplified the global cooperative spirit. While technical advancements were celebrated, the podcast accentuated the persistent policy challenges and the quintessential value of face-to-face interactions in conferences to foster personal and professional relationships in the realm of bioinformatics.

Sorry about the audio quality, we were outdoors and our microphones weren't working as intended.

Ep 9 Update from Global Microbial Identifier

In this episode of the Micro Binfie Podcast, Andrew Page and Lee Katz interview Titus Brown about his journey from studying math and physics as an undergrad to becoming a bioinformatician focusing on metagenomics and software development.

Topics discussed:

Titus' background in math, physics, digital evolution research, and developmental biology
His transition into bioinformatics to analyze the influx of genomic data in the 1990s
Developing early tools for comparative genomics and sequence analysis
The philosophy of creating usable software with good documentation
Work on transcriptomics, metagenomics, and k-mers at Michigan State
Digital normalization and dealing with large sequencing datasets
Moving to UC Davis and continuing work on metagenomics and software like khmer and sourmash
Thoughts on challenges around data reuse and accessibility in science.

Papers:
Spacegraphcats - https://genomebiology.biomedcentral.com/articles/10.1186/s13059-020-02066-4
Sourmash - https://www.biorxiv.org/content/10.1101/2022.01.11.475838v2
IBD exploration - https://dib-lab.github.io/2021-paper-ibd/

117 From Math to Metagenomics - Titus Brown on Career Journeys and Software Solutions

In this episode, Andrew Page and Lee Katz continue their chat with Titus Brown, focusing on taxonomy assignment in metagenomics:

Topics discussed:

Dealing with contamination and low quality genomes in reference databases
Sourmash as a versatile search tool, not a curated database
The need for high confidence in taxonomic assignment in public health
Most microbial assignment tools have low specificity or sensitivity
Possible ways to achieve perfect species classification (in theory)
The challenges around defining species based on small genomic differences
Interesting cryptography concept of 'unicity' distance for classification
Conveying the nuances and uncertainties in taxonomic assignment
The conversation highlights the difficulties around taxonomic classification, especially at the species level, but explores ideas for improving accuracy. Overall it emphasizes the complexities of biology and need for transparent conveyance of uncertainties.

Papers:
Spacegraphcats - https://genomebiology.biomedcentral.com/articles/10.1186/s13059-020-02066-4
Sourmash - https://www.biorxiv.org/content/10.1101/2022.01.11.475838v2
IBD exploration - https://dib-lab.github.io/2021-paper-ibd/

119 The Challenges of Microbial Taxonomy - Conversations with Titus Brown

In this final episode with Titus Brown, the conversation focuses on his work scaling metagenomic search with Sourmash:

An overview of what Sourmash does - sketching and comparing large k-mer datasets
How the sampling approach enables analyses like containment estimation
Exciting capabilities of the Branchwater tool for multi-threaded real-time SRA search
Scaling to search across millions of metagenomes in seconds with WebAssembly
Potential public health applications for tracking and sourcing pathogens
Important caveats around resolution limits and need for follow-up analyses
Ongoing work to characterize the technique's specificity and sensitivity
Overall, this episode highlights the massive scaling Sourmash enables for metagenomic search, and the potential use cases in public health, while acknowledging current limitations and uncertainties. Titus emphasizes the need to precisely convey what bioinformatic tools can and cannot do as research continues.

Papers:
Spacegraphcats - https://genomebiology.biomedcentral.com/articles/10.1186/s13059-020-02066-4
Sourmash - https://www.biorxiv.org/content/10.1101/2022.01.11.475838v2
IBD exploration - https://dib-lab.github.io/2021-paper-ibd/

120 Scaling Metagenomic Search with Sourmash - Conversations with Titus Brown

Join us in this enlightening episode of the Bioinformatics Lab podcast, where we dive deep into the world of metagenomics. In this episode, we explore the complexities and challenges of metagenomics, both from a wet lab and bioinformatics perspective, offering insights into this rapidly evolving field.


🔬 *Key Topics Covered:*
1. *Understanding Metagenomics:* An introduction to the field, highlighting its complexities compared to traditional microbiological techniques.
2. *Techniques in Focus:* Differentiating between amplicon-based sequencing and shotgun metagenomics, and their unique applications.
3. *Navigating Technical Challenges:* Discussing the difficulties in assembling and analyzing diverse DNA mixtures from environmental samples.
4. *Public Health Implications:* Exploring the shift in diagnostic methods in public health labs and how metagenomics is playing a pivotal role.
5. *Innovative Approaches:* Examining emerging techniques like high-throughput multiplexed PCR and their impact on pathogen identification.
6. *Analytical Challenges:* Addressing issues in differentiating between various strains and the challenges of contamination and data interpretation.
7. *The Future of Metagenomics:* Predictions and expectations for the advancement and application of metagenomic techniques in various fields.


🧬 Whether you're a seasoned scientist, a student of microbiology, or simply a science enthusiast, this podcast offers a comprehensive yet accessible discussion on metagenomics. Join us as we unravel the complexities and anticipate the future of this fascinating field.


🎧 *Listen, Learn, and Discover:*
- How metagenomics is reshaping our approach to pathogen identification.
- The technical hurdles faced in the lab and in data analysis.
- The promising future and potential applications of metagenomics in public health and beyond.


📌 *Stay Tuned:* For more insightful discussions and the latest updates in the field of bioinformatics and genomics, subscribe to our channel and hit the bell icon for notifications!


#Metagenomics #Bioinformatics #PublicHealth #Genomics #Microbiology #SciencePodcast #Bioinformaticslab

Ep 17 Metagenomics

Andrew and Lee talk with Christine and Cynney about the Haiti cholera outbreak

Cynney Walters: https://www.linkedin.com/in/cynney-walters-763111190

Walters et al, "Genome sequences from a reemergence of Vibrio cholerae in Haiti, 2022 reveal relatedness to previously circulating strains" https://journals.asm.org/doi/abs/10.1128/jcm.00142-23

128 Haiti cholera outbreak with Christine Lee and Cynney Walters

In this episode of the Micro Binfie Podcast, host Andrew Page catches up with Torsten Seemann at the 10th Microbial Bioinformatics Hackathon in Bethesda, Maryland. They discuss the rapid evolution of bioinformatics, the challenges faced by labs worldwide, and the explosion of tools post-COVID. Torsten shares insights into his work at Melbourne’s Microbiological Diagnostic Unit (MDU), the development of platforms like OzTracker for bacterial genomics, and how his lab plays a national and international role in data sharing. 

The conversation dives into the future of the widely-used variant calling tool Snippy, as Torsten reveals exciting updates funded by the Chan Zuckerberg Initiative, including nanopore read support and the ability to process pre-assembled genomes. They also explore the importance of maintaining open-source bioinformatics tools to prevent them from becoming obsolete. Tune in for an in-depth discussion on the state of genomics, software development, and the challenges and rewards of open-source collaboration.

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Ray COYLE