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Large-scale population based analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short read whole genome sequencing. However, standard short-read approaches, used primarily due to accuracy, throughput and costs, fail to give a complete picture of a genome. They struggle to identify large, balanced structural events, cannot access repetitive regions of the genome and fail to resolve the human genome into its two haplotypes. Here we describe an approach that retains long range information while harnessing the advantages of short reads. Starting from only~ ng of DNA, we produce barcoded short read libraries. The use of novel informatic approaches allows for the barcoded short reads to be associated with the long molecules of origin producing a novel datatype known as 'Linked-Reads'. This approach allows for simultaneous detection of small and large variants from a single Linked-Read library. We have previously demonstrated the utility of whole genome Linked-Reads (lrWGS) for performing diploid, de novo assembly of individual genomes (Weisenfeld et al. ). In this manuscript, weshow the advantages of Linked-Reads over standard short read approaches for reference based analysis. We demonstrate the ability of Linked-Reads to reconstruct megabase scale haplotypes and to recover parts of the genome that are typically inaccessible to short reads, including phenotypically important genes such as STRC, SMN and SMN . We demonstrate the ability of both lrWGS and Linked-Read Whole Exome Sequencing (lrWES) to identify complex structural variations, including balanced events, single exon deletions, and single exon duplications. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

Large-scale population analyses coupled with advances in technology have demonstrated that the human genome is more diverse than originally thought. To date, this diversity has largely been uncovered using short-read whole-genome sequencing. However, these short-read approaches fail to give a complete picture of a genome. They struggle to identify structural events, cannot access repetitive regions, and fail to resolve the human genome into haplotypes. Here, we describe an approach that retains long range information while maintaining the advantages of short reads. Starting from ∼1 ng of high molecular weight DNA, we produce barcoded short-read libraries. Novel informatic approaches allow for the barcoded short reads to be associated with their original long molecules producing a novel data type known as "Linked-Reads". This approach allows for simultaneous detection of small and large variants from a single library. In this manuscript, we show the advantages of Linked-Reads over standard short-read approaches for reference-based analysis. Linked-Reads allow mapping to 38 Mb of sequence not accessible to short reads, adding sequence in 423 difficult-to-sequence genes including disease-relevant genes STRC, SMN1, and SMN2. Both Linked-Read whole-genome and whole-exome sequencing identify complex structural variations, including balanced events and single exon deletions and duplications. Further, Linked-Reads extend the region of high-confidence calls by 68.9 Mb. The data presented here show that Linked-Reads provide a scalable approach for comprehensive genome analysis that is not possible using short reads alone.

The Ll.LtrB intron from the Gram-positive bacterium Lactococcus lactis is one of the most studied bacterial group II introns. Ll.LtrB interrupts the relaxase gene of three L. lactis conjugative elements. The relaxase enzyme recognizes the origin of transfer (oriT ) and initiates the intercellular transfer of its conjugative element. The splicing efficiency of Ll.LtrB from the relaxase transcript thus controls the conjugation level of its host element. Here, we used the level of sex factor conjugation as a read-out for Ll.LtrB splicing efficiency. Using this highly sensitive splicing/conjugation assay (107-fold detection range), we demonstrate that Ll.LtrB can trans-splice in L. lactis when fragmented at various positions such as: three different locations within domain IV, within domain I and within domain III. We also demonstrate that the intron-encoded protein, LtrA, is absolutely required for Ll.LtrB trans-splicing. Characteristic Y-branched trans-spliced introns and ligated exons are detected by RT-PCR from total RNA extracts of cells harbouring fragmented Ll.LtrB. The splicing/conjugation assay we developed constitutes the first model system to study group II intron trans-splicing in vivo. Although only previously observed in bacterial-derived organelles, we demonstrate that assembly and trans-splicing of a fragmented group II intron can take place efficiently in bacterial cells.

SummarySome self-splicing group II introns (ribozymes) are mobile retroelements. These retroelements, which can insert themselves into cognate intronless alleles or ectopic sites by reverse splicing, are thought to be the evolutionary progenitors of the widely distributed eukaryotic spliceosomal introns. Lateral or horizontal transmission of introns (i.e. between species), although never experimentally demonstrated, is a well-accepted model for intron dispersal and evolution. Horizontal transfer of the ancestral bacterial group II introns may have contributed to the dispersal and wide distribution of spliceosomal introns present in modern eukaryotic genomes. Here, the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis was used as a model system to address the dissemination of introns in the bacterial kingdom. We report the first experimental demonstration of horizontal transfer of a group II intron. We show that the Ll.LtrB group II intron, originally discovered on an L. lactis conjugative plasmid (pRS01) and within a chromosomally located sex factor in L. lactis 712, invades new sites using both retrohoming and retrotransposition pathways after its transfer by conjugation. Ll.LtrB lateral transfer is shown among different L. lactis strains (intraspecies) (retrohoming and retrotransposition) and between L. lactis and Enterococcus faecalis (interspecies) (retrohoming). These results shed light on long-standing questions about intron evolution and propagation, and demonstrate that conjugation is one of the mechanisms by which group II introns are, and probably were, broadly disseminated between widely diverged organisms.

Group II introns are found in organelles, bacteria, and archaea. Some harbor an open reading frame (ORF) with reverse transcriptase, maturase, and occasionally endonuclease activities. Group II introns require the assistance of either intronencoded or free-standing maturases to excise from primary RNA transcripts in vivo. Some ORF-containing group II introns were shown to be mobile retroelements that invade new DNA sites by retrohoming or retrotransposition. Group II introns are also hypothesized to be the ancestors of the spliceosome-dependent nuclear introns and the small nuclear RNAs (snRNAs-U1, U2, U4, U5, and U6) that are part of the spliceosome. The ability of some fragmented group II introns to undergo splicing in trans supports the theory that the snRNAs evolved from portions of group II introns. Here, we developed a Tn5-based genetic screen to explore the trans-splicing potential of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis. Proficient trans-splicing variants of Ll.LtrB were selected using a highly sensitive trans-splicing/conjugation screen. We report that numerous fragmentation sites located throughout Ll.LtrB support splicing in trans, showing that this intron is remarkably more tolerant to fragmentation than expected from the fragmentation sites uncovered within natural trans-splicing group II introns. This work unveils the great versatility of group II intron fragments to assemble and accurately trans-splice their flanking exons in vivo.

The intracellular bacterium Francisella tularensis is the causative agent of tularemia, a potentially fatal disease. In macrophages, Francisella escapes the initial phagosome and replicates in the cytosol, where it is detected by the cytosolic DNA sensor AIM2 leading to activation of the AIM2 inflammasome. However, during aerosol infection, Francisella is also taken up by dendritic cells. In this study, we show that Francisella novicida escapes into the cytosol of bone marrow-derived dendritic cells (BMDC) where it undergoes rapid replication. We show that F. novicida activates the AIM2 inflammasome in BMDC, causing release of large amounts of IL-1β and rapid host cell death. The Francisella Pathogenicity Island is required for bacterial escape and replication and for inflammasome activation in dendritic cells. In addition, we show that bacterial DNA is bound by AIM2, which leads to inflammasome assembly in infected dendritic cells. IFN-β is upregulated in BMDC following Francisella infection, and the IFN-β signaling pathway is partially required for inflammasome activation in this cell type. Taken together, our results demonstrate that F. novicida induces inflammasome activation in dendritic cells. The resulting inflammatory cell death may be beneficial to remove the bacterial replicative niche and protect the host.