July 7, 2019  |  

Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain.Although in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S.Our study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A. phagocytophilum.

July 7, 2019  |  

Strobe sequence design for haplotype assembly.

Humans are diploid, carrying two copies of each chromosome, one from each parent. Separating the paternal and maternal chromosomes is an important component of genetic analyses such as determining genetic association, inferring evolutionary scenarios, computing recombination rates, and detecting cis-regulatory events. As the pair of chromosomes are mostly identical to each other, linking together of alleles at heterozygous sites is sufficient to phase, or separate the two chromosomes. In Haplotype Assembly, the linking is done by sequenced fragments that overlap two heterozygous sites. While there has been a lot of research on correcting errors to achieve accurate haplotypes via assembly, relatively little work has been done on designing sequencing experiments to get long haplotypes. Here, we describe the different design parameters that can be adjusted with next generation and upcoming sequencing technologies, and study the impact of design choice on the length of the haplotype.We show that a number of parameters influence haplotype length, with the most significant one being the advance length (distance between two fragments of a clone). Given technologies like strobe sequencing that allow for large variations in advance lengths, we design and implement a simulated annealing algorithm to sample a large space of distributions over advance-lengths. Extensive simulations on individual genomic sequences suggest that a non-trivial distribution over advance lengths results a 1-2 order of magnitude improvement in median haplotype length.Our results suggest that haplotyping of large, biologically important genomic regions is feasible with current technologies.

July 7, 2019  |  

Structural variation analysis with strobe reads.

Structural variation including deletions, duplications and rearrangements of DNA sequence are an important contributor to genome variation in many organisms. In human, many structural variants are found in complex and highly repetitive regions of the genome making their identification difficult. A new sequencing technology called strobe sequencing generates strobe reads containing multiple subreads from a single contiguous fragment of DNA. Strobe reads thus generalize the concept of paired reads, or mate pairs, that have been routinely used for structural variant detection. Strobe sequencing holds promise for unraveling complex variants that have been difficult to characterize with current sequencing technologies.We introduce an algorithm for identification of structural variants using strobe sequencing data. We consider strobe reads from a test genome that have multiple possible alignments to a reference genome due to sequencing errors and/or repetitive sequences in the reference. We formulate the combinatorial optimization problem of finding the minimum number of structural variants in the test genome that are consistent with these alignments. We solve this problem using an integer linear program. Using simulated strobe sequencing data, we show that our algorithm has better sensitivity and specificity than paired read approaches for structural variation identification.braphael@brown.edu

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