FUNCTIONALLY PROFILING METAGENOMES AND... - Eric Franzosa - Late-Breaking Research - ISMB 2016

Summary

In the script, Dr. Orlando discusses advancements in profiling microbial communities from sequencing data, focusing on taxonomic and functional profiling. The tool 'MetaPhlan 2' is highlighted for taxonomic profiling, while 'HUMAnN2' is introduced for functional profiling, using a tiered read mapping strategy to improve accuracy and speed. HUMAnN2 enables species-level functional profiling, offering insights into both the composition and activity of microbial communities, with applications demonstrated in the Human Microbiome Project and other cohorts.

Takeaways

• 🌟 The script discusses advancements in profiling microbial communities from shotgun sequencing data, focusing on taxonomic and functional profiling.
• 🔍 Taxonomic profiling identifies species and higher-level clades present in a microbial community, while functional profiling identifies gene families and pathway composition.
• 🚀 MetaPhlan 2 is a tool developed for taxonomic profiling that searches reads against a pre-selected set of marker genes unique to each clade or species for efficiency and accuracy.
• 🛠 Humann2 introduces a 'tiered read mapping' strategy to improve the accuracy, speed, and resolution of functional profiling by leveraging species identification for more targeted searches.
• 🔑 The first tier of Humann2's method rapidly identifies species in a community by mapping reads against species-specific marker genes.
• 🌐 The second tier builds a custom database for the sample by concatenating the pan-genomes of detected species, allowing for a detailed search of the remaining reads against these genomes.
• 📚 Humann2's tiered search is more specific and sensitive, leading to higher accuracy and faster processing times compared to traditional comprehensive searches.
• 📈 The method was tested using synthetic metagenomes created from sets of bacterial genomes, demonstrating high sensitivity and precision even for low-abundance species.
• 🌿 Humann2 has been applied to real-world data, profiling hundreds of human metagenomes, and has shown to explain a majority of reads during the initial accelerated search tier.
• 🔬 The tool allows for the identification of metabolic pathways that are signatures for particular body areas, providing insights into the taxonomic resolution of functions within communities.
• 📊 Humann2 also enables the distinction between functional potential (DNA level) and functional activity (RNA level), showing that the two are not always the same.

Q & A

What are the two primary questions in microbial community profiling from sequencing data?

The two primary questions are 'who is there', which refers to taxonomic profiling to identify species and higher-level clades, and 'what are those species doing', which is functional profiling to identify gene families and pathway composition.

What is the computational challenge in profiling microbial communities from sequencing data?

The computational challenge lies in the need to search short reads from shotgun sequencing against a vast database of microbial reference genomes, which is computationally intensive and prone to errors due to potential spurious mappings.

What is MetaPhlAn 2 and how does it address the taxonomic profiling challenge?

MetaPhlAn 2 is a tool developed for taxonomic profiling that searches reads against a pre-selected set of marker genes unique to each clade or species, rather than the entire database, making the profiling process more efficient and accurate.

What is the concept behind the tool HUMAn2 and its tiered read mapping strategy?

HUMAn2 implements a tiered read mapping strategy, which starts with rapidly identifying species in a community by mapping reads against species-specific marker genes, followed by a custom database search of pan genomes of detected species, and finally a comprehensive search for any remaining reads.

How does HUMAn2 improve the accuracy and speed of functional profiling?

HUMAn2 improves accuracy by ensuring reads are mapped to the correct species-specific genes, reducing spurious mappings. It improves speed by using a reduced database for the initial search, thus processing the metagenome much faster than a comprehensive search against an exhaustive database.

What is the advantage of HUMAn2's tiered search over traditional comprehensive searches?

HUMAn2's tiered search is more specific and sensitive, placing reads in the correct locations, and thus increasing overall accuracy. It also processes the metagenome faster due to the use of a reduced pan genome database.

How does HUMAn2 provide species-level resolution in functional profiling?

HUMAn2's tiered search allows for the reconstruction of functions on a species-by-species basis within the community, providing insights into which species are performing specific functions, even for low-abundance species.

What synthetic metagenomes or meta transcriptomes are used to evaluate HUMAn2's performance?

Synthetic metagenomes or meta transcriptomes are created by taking sets of bacterial genomes and pulling synthetic sequencing reads from them, allowing for the evaluation of HUMAn2's accuracy and performance against expected profiles.

What are the different patterns of species contribution to a conserved pathway observed in the human gut microbiome using HUMAn2?

Different patterns include a complex attribution where multiple species contribute to a conserved pathway in varying mixtures across individuals, a per-person dominant attribution where one species dominates the contribution to the pathway for each individual, and a universal dominant pattern where one species consistently provides the pathway across the population.

How does HUMAn2 distinguish between functional potential and functional activity in a community?

HUMAn2 can analyze both DNA and RNA data, allowing it to distinguish between the functional potential (number of encoded pathways) and functional activity (actual expression of those pathways) within a community, showing that the two are not always the same.

How can one access and learn more about HUMAn2 and its related tools?

HUMAn2 can be found by searching its name online, and it is installable via source pip as a Python package and via Homebrew. There is a detailed user manual and an active user group on Google Groups for further support and information.