Targeted High-Resolution Taxonomic Identification of Bifidobacterium longum subsp. infantis Using Human Milk Oligosaccharide Metabolizing Genes.

Bifidobacterium longum subsp. infantis (B. infantis) is one of a few microorganisms capable of metabolizing human breast milk and is a pioneer colonizer in the guts of breastfed infants. One current challenge is differentiating B. infantis from its close relatives, B. longum and B. suis. All three organisms are classified in the same species group but only B. infantis can metabolize human milk oligosaccharides (HMOs). We compared HMO-metabolizing genes across different Bifidobacterium genomes and developed B. infantis-specific primers to determine if the genes alone or the primers can be used to quickly characterize B. infantis. We showed that B. infantis is uniquely identified by the presence of five HMO-metabolizing gene clusters, tested for its prevalence in infant gut metagenomes, and validated the results using the B. infantis-specific primers. We observed that only 15 of 203 (7.4%) children under 2 years old from a cohort of US children harbored B. infantis. These results highlight the importance of developing and improving approaches to identify B. infantis. A more accurate characterization may provide insights into regional differences of B. infantis prevalence in infant gut microbiota.

CRISPAltRations: a validated cloud-based approach for interrogation of double-strand break repair mediated by CRISPR genome editing.

CRISPR systems enable targeted genome editing in a wide variety of organisms by introducing single- or double-strand DNA breaks, which are repaired using endogenous molecular pathways. Characterization of on- and off-target editing events from CRISPR proteins can be evaluated using targeted genome resequencing. We characterized DNA repair fingerprints that result from non-homologous end joining (NHEJ) after double-stranded breaks (DSBs) were introduced by Cas9 or Cas12a for >500 paired treatment/control experiments. We found that building biological understanding of the repair into a novel analysis tool (CRISPAltRations) improved the quality of the results. We validated our software using simulated, targeted amplicon sequencing data (11 guide RNAs [gRNAs] and 603 on- and off-target locations) and demonstrated that CRISPAltRations outperforms other publicly available software tools in accurately annotating CRISPR-associated indels and homology-directed repair (HDR) events. We enable non-bioinformaticians to use CRISPAltRations by developing a web-accessible, cloud-hosted deployment, which allows rapid batch processing of samples in a graphical user interface (GUI) and complies with HIPAA security standards. By ensuring that our software is thoroughly tested, version controlled, and supported with a user interface (UI), we enable resequencing analysis of CRISPR genome editing experiments to researchers no matter their skill in bioinformatics.