ProtoCode: Leveraging large language models (LLMs) for automated generation of machine-readable PCR protocols from scientific publications.

Protocol standardization and sharing are crucial for reproducibility in life sciences. In spite of numerous efforts for standardized protocol description, adherence to these standards in literature remains largely inconsistent. Curation of protocols are especially challenging due to the labor intensive process, requiring expert domain knowledge of each experimental procedure. Recent advancements in Large Language Models (LLMs) offer a promising solution to interpret and curate knowledge from complex scientific literature. In this work, we develop ProtoCode, a tool leveraging fine-tune LLMs to curate protocols into intermediate representation formats which can be interpretable by both human and machine interfaces. Our proof-of-concept, focused on polymerase chain reaction (PCR) protocols, retrieves information from PCR protocols at an accuracy ranging 69-100 % depending on the information content. In all tested protocols, we demonstrate that ProtoCode successfully converts literature-based protocols into correct operational files for multiple thermal cycler systems. In conclusion, ProtoCode can alleviate labor intensive curation and standardization of life science protocols to enhance research reproducibility by providing a reliable, automated means to process and standardize protocols. ProtoCode is freely available as a web server at https://curation.taxila.io/ProtoCode/.

Parallel Nonfunctionalization of CK1δ/ε Kinase Ohnologs Following a Whole-Genome Duplication Event.

Whole-genome duplication (WGD) followed by speciation allows us to examine the parallel evolution of ohnolog pairs. In the yeast family Saccharomycetaceae, HRR25 is a rare case of repeated ohnolog maintenance. This gene has reverted to a single copy in Saccharomyces cerevisiae where it is now essential, but has been maintained as pairs in at least 7 species post-WGD. In S. cerevisiae, HRR25 encodes the casein kinase 1δ/ε and plays a role in a variety of functions through its kinase activity and protein-protein interactions (PPIs). We hypothesized that the maintenance of duplicated HRR25 ohnologs could be a result of repeated subfunctionalization. We tested this hypothesis through a functional complementation assay in S. cerevisiae, testing all pairwise combinations of 25 orthologs (including 7 ohnolog pairs). Contrary to our expectations, we observed no cases of pair-dependent complementation, which would have supported the subfunctionalization hypothesis. Instead, most post-WGD species have one ohnolog that failed to complement, suggesting their nonfunctionalization or neofunctionalization. The ohnologs incapable of complementation have undergone more rapid protein evolution, lost most PPIs that were observed for their functional counterparts and singletons from post-WGD and non-WGD species, and have nonconserved cellular localization, consistent with their ongoing loss of function. The analysis in Naumovozyma castellii shows that the noncomplementing ohnolog is expressed at a lower level and has become nonessential. Taken together, our results indicate that HRR25 orthologs are undergoing gradual nonfunctionalization.

Parallel nonfunctionalization of CK1δ/ε kinase ohnologs following a whole-genome duplication event.

Whole genome duplication (WGD) followed by speciation allows us to examine the parallel evolution of ohnolog pairs. In the yeast family Saccharomycetaceae, HRR25 is a rare case of repeated ohnolog maintenance. This gene has reverted to a single copy in S. cerevisiae where it is now essential, but has been maintained as pairs in at least 7 species post WGD. In S. cerevisiae, HRR25 encodes the casein kinase (CK) 1δ/ε and plays a role in a variety of functions through its kinase activity and protein-protein interactions (PPIs). We hypothesized that the maintenance of duplicated HRR25 ohnologs could be a result of repeated subfunctionalization. We tested this hypothesis through a functional complementation assay in S. cerevisiae, testing all pairwise combinations of 25 orthologs (including 7 ohnolog pairs). Contrary to our expectations, we observed no cases of pair-dependent complementation, which would have supported the subfunctionalization hypothesis. Instead, most post-WGD species have one ohnolog that failed to complement, suggesting their nonfunctionalization or neofunctionalization. The ohnologs incapable of complementation have undergone more rapid protein evolution, lost most PPIs that were observed for their functional counterparts and singletons from post and non-WGD species, and have non-conserved cellular localization, consistent with their ongoing loss of function. The analysis in N. castelli shows that the non-complementing ohnolog is expressed at a lower level and has become non-essential. Taken together, our results indicate that HRR25 orthologs are undergoing gradual nonfunctionalization.

A universal sequencing read interpreter.

Massively parallel DNA sequencing has led to the rapid growth of highly multiplexed experiments in biology. These experiments produce unique sequencing results that require specific analysis pipelines to decode highly structured reads. However, no versatile framework that interprets sequencing reads to extract their encoded information for downstream biological analysis has been developed. Here, we report INTERSTELLAR (interpretation, scalable transformation, and emulation of large-scale sequencing reads) that decodes data values encoded in theoretically any type of sequencing read and translates them into sequencing reads of another structure of choice. We demonstrated that INTERSTELLAR successfully extracted information from a range of short- and long-read sequencing reads and translated those of single-cell (sc)RNA-seq, scATAC-seq, and spatial transcriptomics to be analyzed by different software tools that have been developed for conceptually the same types of experiments. INTERSTELLAR will greatly facilitate the development of sequencing-based experiments and sharing of data analysis pipelines.

Barcode fusion genetics-protein-fragment complementation assay (BFG-PCA): tools and resources that expand the potential for binary protein interaction discovery.

Barcode fusion genetics (BFG) utilizes deep sequencing to improve the throughput of protein-protein interaction (PPI) screening in pools. BFG has been implemented in Yeast two-hybrid (Y2H) screens (BFG-Y2H). While Y2H requires test protein pairs to localize in the nucleus for reporter reconstruction, dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA) allows proteins to localize in broader subcellular contexts and proves to be largely orthogonal to Y2H. Here, we implemented BFG to DHFR-PCA (BFG-PCA). This plasmid-based system can leverage ORF collections across model organisms to perform comparative analysis, unlike the original DHFR-PCA that requires yeast genomic integration. The scalability and quality of BFG-PCA were demonstrated by screening human and yeast interactions for >11 000 bait-prey pairs. BFG-PCA showed high-sensitivity and high-specificity for capturing known interactions for both species. BFG-Y2H and BFG-PCA capture distinct sets of PPIs, which can partially be explained based on the domain orientation of the reporter tags. BFG-PCA is a high-throughput protein interaction technology to interrogate binary PPIs that exploits clone collections from any species of interest, expanding the scope of PPI assays.

Machine learning approach for discrimination of genotypes based on bright-field cellular images.

Morphological profiling is a combination of established optical microscopes and cutting-edge machine vision technologies, which stacks up successful applications in high-throughput phenotyping. One major question is how much information can be extracted from an image to identify genetic differences between cells. While fluorescent microscopy images of specific organelles have been broadly used for single-cell profiling, the potential ability of bright-field (BF) microscopy images of label-free cells remains to be tested. Here, we examine whether single-gene perturbation can be discriminated based on BF images of label-free cells using a machine learning approach. We acquired hundreds of BF images of single-gene mutant cells, quantified single-cell profiles consisting of texture features of cellular regions, and constructed a machine learning model to discriminate mutant cells from wild-type cells. Interestingly, the mutants were successfully discriminated from the wild type (area under the receiver operating characteristic curve = 0.773). The features that contributed to the discrimination were identified, and they included those related to the morphology of structures that appeared within cellular regions. Furthermore, functionally close gene pairs showed similar feature profiles of the mutant cells. Our study reveals that single-gene mutant cells can be discriminated from wild-type cells based on BF images, suggesting the potential as a useful tool for mutant cell profiling.

Complete Genome Sequence of Bacillus sp. Strain KH172YL63, Isolated from Deep-Sea Sediment.

Bacillus sp. strain KH172YL63 is a Gram-positive bacterium isolated from the deep-sea floor surface sediment at 3,308 m below sea level in the Nankai Trough in Japan. Here, we report the complete genome sequence of Bacillus sp. strain KH172YL63, which has a genome size of 4,251,700 bp and a G+C content of 44.8%.

The role of structural pleiotropy and regulatory evolution in the retention of heteromers of paralogs.

Gene duplication is a driver of the evolution of new functions. The duplication of genes encoding homomeric proteins leads to the formation of homomers and heteromers of paralogs, creating new complexes after a single duplication event. The loss of these heteromers may be required for the two paralogs to evolve independent functions. Using yeast as a model, we find that heteromerization is frequent among duplicated homomers and correlates with functional similarity between paralogs. Using in silico evolution, we show that for homomers and heteromers sharing binding interfaces, mutations in one paralog can have structural pleiotropic effects on both interactions, resulting in highly correlated responses of the complexes to selection. Therefore, heteromerization could be preserved indirectly due to selection for the maintenance of homomers, thus slowing down functional divergence between paralogs. We suggest that paralogs can overcome the obstacle of structural pleiotropy by regulatory evolution at the transcriptional and post-translational levels.

Complete Genome Sequence of Psychrobacter sp. Strain KH172YL61, Isolated from Deep-Sea Sediments in the Nankai Trough, Japan.

Psychrobacter sp. strain KH172YL61 is a Gram-negative bacterium isolated from deep-sea sediment in the Nankai Trough in Japan. Here, we report the complete genome sequence of this strain, which has a genome size of 3.19 Mb, with a G+C content of 44.0%.

Fast and global detection of periodic sequence repeats in large genomic resources.

Periodically repeating DNA and protein elements are involved in various important biological events including genomic evolution, gene regulation, protein complex formation, and immunity. Notably, the currently used genome editing tools such as ZFNs, TALENs, and CRISPRs are also all associated with periodically repeating biomolecules of natural organisms. Despite the biological importance of periodically repeating sequences and the expectation that new genome editing modules could be discovered from such periodical repeats, no software that globally detects such structured elements in large genomic resources in a high-throughput and unsupervised manner has been developed. We developed new software, SPADE (Search for Patterned DNA Elements), that exhaustively explores periodic DNA and protein repeats from large-scale genomic datasets based on k-mer periodicity evaluation. With a simple constraint, sequence periodicity, SPADE captured reported genome-editing-associated sequences and other protein families involving repeating domains such as tetratricopeptide, ankyrin and WD40 repeats with better performance than the other software designed for limited sets of repetitive biomolecular sequences, suggesting the high potential of this software to contribute to the discovery of new biological events and new genome editing modules.