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Asset Tag: Targeted sequencing,

July 19, 2019

Radical remodeling of the Y chromosome in a recent radiation of malaria mosquitoes.

Y chromosomes control essential male functions in many species, including sex determination and fertility. However, because of obstacles posed by repeat-rich heterochromatin, knowledge of Y chromosome sequences is limited to a handful of model organisms, constraining our understanding of Y biology across the tree of life. Here, we leverage long single-molecule sequencing to determine the content and structure of the nonrecombining Y chromosome of the primary African malaria mosquito, Anopheles gambiae. We find that the An. gambiae Y consists almost entirely of a few massively amplified, tandemly arrayed repeats, some of which can recombine with similar repeats on the X chromosome. Sex-specific genome resequencing in a recent species radiation, the An. gambiae complex, revealed rapid sequence turnover within An. gambiae and among species. Exploiting 52 sex-specific An. gambiae RNA-Seq datasets representing all developmental stages, we identified a small repertoire of Y-linked genes that lack X gametologs and are not Y-linked in any other species except An. gambiae, with the notable exception of YG2, a candidate male-determining gene. YG2 is the only gene conserved and exclusive to the Y in all species examined, yet sequence similarity to YG2 is not detectable in the genome of a more distant mosquito relative, suggesting rapid evolution of Y chromosome genes in this highly dynamic genus of malaria vectors. The extensive characterization of the An. gambiae Y provides a long-awaited foundation for studying male mosquito biology, and will inform novel mosquito control strategies based on the manipulation of Y chromosomes.

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July 19, 2019

Comprehensive mutagenesis of the fimS promoter regulatory switch reveals novel regulation of type 1 pili in uropathogenic Escherichia coli.

Type 1 pili (T1P) are major virulence factors for uropathogenic Escherichia coli (UPEC), which cause both acute and recurrent urinary tract infections. T1P expression therefore is of direct relevance for disease. T1P are phase variable (both piliated and nonpiliated bacteria exist in a clonal population) and are controlled by an invertible DNA switch (fimS), which contains the promoter for the fim operon encoding T1P. Inversion of fimS is stochastic but may be biased by environmental conditions and other signals that ultimately converge at fimS itself. Previous studies of fimS sequences important for T1P phase variation have focused on laboratory-adapted E. coli strains and have been limited in the number of mutations or by alteration of the fimS genomic context. We surmounted these limitations by using saturating genomic mutagenesis of fimS coupled with accurate sequencing to detect both mutations and phase status simultaneously. In addition to the sequences known to be important for biasing fimS inversion, our method also identifies a previously unknown pair of 5′ UTR inverted repeats that act by altering the relative fimA levels to control phase variation. Thus we have uncovered an additional layer of T1P regulation potentially impacting virulence and the coordinate expression of multiple pilus systems.

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July 19, 2019

Polymerase specific error rates and profiles identified by single molecule sequencing.

DNA polymerases have an innate error rate which is polymerase and DNA context specific. Historically the mutational rate and profiles have been measured using a variety of methods, each with their own technical limitations. Here we used the unique properties of single molecule sequencing to evaluate the mutational rate and profiles of six DNA polymerases at the sequence level. In addition to accurately determining mutations in double strands, single molecule sequencing also captures direction specific transversions and transitions through the analysis of heteroduplexes. Not only did the error rates vary, but also the direction specific transitions differed among polymerases. Copyright © 2016 Elsevier B.V. All rights reserved.

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July 19, 2019

Mitotic intragenic recombination: A mechanism of survival for several congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDGs) are disorders of abnormal protein glycosylation that affect multiple organ systems. Because most CDGs have been described in only a few individuals, our understanding of the associated phenotypes and the mechanisms of individual survival are limited. In the process of studying two siblings, aged 6 and 11 years, with MOGS-CDG and biallelic MOGS (mannosyl-oligosaccharide glucosidase) mutations (GenBank: NM_006302.2; c.[65C>A; 329G>A] p.[Ala22Glu; Arg110His]; c.[370C>T] p.[Gln124(*)]), we noted that their survival was much longer than the previous report of MOGS-CDG, in a child who died at 74 days of age. Upon mutation analysis, we detected multiple MOGS genotypes including wild-type alleles in their cultured fibroblast and peripheral blood DNA. Further analysis of DNA from cultured fibroblasts of six individuals with compound heterozygous mutations of PMM2 (PMM2-CDG), MPI (MPI-CDG), ALG3 (ALG3-CDG), ALG12 (ALG12-CDG), DPAGT1 (DPAGT1-CDG), and ALG1 (ALG1-CDG) also identified multiple genotypes including wild-type alleles for each. Droplet digital PCR showed a ratio of nearly 1:1 wild-type to mutant alleles for most, but not all, mutations. This suggests that mitotic recombination contributes to the survival and the variable expressivity of individuals with compound heterozygous CDGs. This also provides an explanation for prior observations of a reduced frequency of homozygous mutations and might contribute to increased levels of residual enzyme activity in cultured fibroblasts of individuals with MPI- and PMM2-CDGs. Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

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July 19, 2019

Bats may eat diurnal flies that rest on wind turbines

Bats are currently killed in large numbers at wind turbines worldwide, but the ultimate reason why this happens remains poorly understood. One hypothesis is that bats visit wind turbines to feed on insects exposed at the turbine towers. We used single molecule next generation DNA sequencing to identify stomach contents of 18 bats of four species (Pipistrellus pygmaeus, Nyctalus noctula, Eptesicus nilssonii and Vespertilio murinus) found dead under wind turbines in southern Sweden. Stomach contents were diverse but included typically diurnal flies, e.g. blow-flies (Calliphoridae), flesh-flies (Sarcophagidae) and houseflies (Muscidae) and also several flightless taxa. Such prey items were eaten by all bat species and at all wind turbine localities and it seems possible that they had been captured at or near the surface of the turbines at night. Using sticky traps, we documented an abundance of swarming (diurnal) ants (Myrmica spp.) and sometimes blow-flies and houseflies at the nacelle house. Near the base of the tower the catches were more diverse and corresponded better with the taxa found in the bat stomachs, including various diurnal flies. To evaluate if flies and other insects resting on the surface of a wind turbine are available to bats, we ensonified a house fly (Musca) on a smooth (plastic) surface with synthetic ultrasonic pulses of the frequencies used by the bat species that we had sampled. The experiment revealed potentially useful echoes, provided the attack angle was low and the frequency high (50–75 kHz). Hence resting flies and other arthropods can probably be detected by echolocating bats on the surface of a wind turbine. Our findings are consistent with published observations of the behavior of bats at wind turbines and may actually explain the function of some of these behaviors.

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July 19, 2019

Nested Russian doll-like genetic mobility drives rapid dissemination of the Carbapenem resistance gene blaKPC

The recent widespread emergence of carbapenem resistance in Enterobacteriaceae is a major public health concern, as carbapenems are a therapy of last resort against this family of common bacterial pathogens. Resistance genes can mobilize via various mechanisms, including conjugation and transposition; however, the importance of this mobility in short-term evolution, such as within nosocomial outbreaks, is unknown. Using a combination of short- and long-read whole-genome sequencing of 281 blaKPC-positive Enterobacteriaceae isolates from a single hospital over 5 years, we demonstrate rapid dissemination of this carbapenem resistance gene to multiple species, strains, and plasmids. Mobility of blaKPC occurs at multiple nested genetic levels, with transmission of blaKPC strains between individuals, frequent transfer of blaKPC plasmids between strains/species, and frequent transposition of blaKPC transposon Tn4401 between plasmids. We also identify a common insertion site for Tn4401 within various Tn2-like elements, suggesting that homologous recombination between Tn2-like elements has enhanced the spread of Tn4401 between different plasmid vectors. Furthermore, while short-read sequencing has known limitations for plasmid assembly, various studies have attempted to overcome this by the use of reference-based methods. We also demonstrate that, as a consequence of the genetic mobility observed in this study, plasmid structures can be extremely dynamic, and therefore these reference-based methods, as well as traditional partial typing methods, can produce very misleading conclusions. Overall, our findings demonstrate that nonclonal resistance gene dissemination can be extremely rapid, presenting significant challenges for public health surveillance and achieving effective control of antibiotic resistance. Copyright © 2016 Sheppard et al.

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July 19, 2019

Accelerated cloning of a potato late blight-resistance gene using RenSeq and SMRT sequencing.

Global yields of potato and tomato crops have fallen owing to potato late blight disease, which is caused by Phytophthora infestans. Although most commercial potato varieties are susceptible to blight, many wild potato relatives show variation for resistance and are therefore a potential source of Resistance to P. infestans (Rpi) genes. Resistance breeding has exploited Rpi genes from closely related tuber-bearing potato relatives, but is laborious and slow. Here we report that the wild, diploid non-tuber-bearing Solanum americanum harbors multiple Rpi genes. We combine resistance (R) gene sequence capture (RenSeq) with single-molecule real-time (SMRT) sequencing (SMRT RenSeq) to clone Rpi-amr3i. This technology should enable de novo assembly of complete nucleotide-binding, leucine-rich repeat receptor (NLR) genes, their regulatory elements and complex multi-NLR loci from uncharacterized germplasm. SMRT RenSeq can be applied to rapidly clone multiple R genes for engineering pathogen-resistant crops.

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July 19, 2019

Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance.

The Bacillus thuringiensis d-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd?=?11-41?nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.

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July 19, 2019

SMRT RenSeq protocol

R gene enrichment and Sequencing (RenSeq, Jupe et al. 2013) is a genome complexity reduction method which allows to enrich for nucleotide-binding, leucine reach repeat (NLR) type plant disease resistance genes prior to sequencing. RenSeq was established and successfully used with Illumina platforms (Jupe et al. 2013, Andolfo et al. 2014), however the repetitive nature of NLR genes hampered de novo assembly of this family. Here we describe a protocol which enables to prepare long enriched libraries that are suitable for Pacific Biosciences Single-Molecule Real Time (SMRT) sequencing. Reads Of Inserts (ROI) generated with this protocol are around 3-4 kb in length (longer than the average NLR sequence). These long reads are especially well suited for de novo assembly of whole NLR genes including their regulatory elements

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July 19, 2019

Large deletions at the SHOX locus in the pseudoautosomal region are associated with skeletal atavism in Shetland ponies.

Skeletal atavism in Shetland ponies is a heritable disorder characterized by abnormal growth of the ulna and fibula that extend the carpal and tarsal joints, respectively. This causes abnormal skeletal structure, impaired movements, and affected foals are usually euthanized. In order to identify the causal mutation we subjected six confirmed Swedish cases and a DNA pool consisting of 21 control individuals to whole genome resequencing. We screened for polymorphisms where the cases and the control pool were fixed for opposite alleles and observed this signature for only 25 SNPs, most of which were scattered on genome assembly unassigned scaffolds. Read depth analysis at these loci revealed homozygosity or compound heterozygosity for two partially overlapping large deletions in the pseudoautosomal region (PAR) of chromosome X/Y in cases but not in the control pool. One of these deletions removes the entire coding region of the SHOX gene and both deletions remove parts of the CRLF2 gene located downstream of SHOX. The horse reference assembly of the PAR is highly fragmented, and in order to characterize this region we sequenced bacterial artificial chromosome (BAC) clones by single-molecule real-time (SMRT) sequencing technology. This considerably improved the assembly and enabled size estimations of the two deletions to 160-180 kb and 60-80 kb, respectively. Complete association between the presence of these deletions and disease status was verified in eight other affected horses. The result of the present study is consistent with previous studies in humans showing crucial importance of SHOX for normal skeletal development. Copyright © 2016 Author et al.

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July 19, 2019

Comparative analyses of low, medium and high-resolution HLA typing technologies for human populations

Human Leukocyte Antigen (HLA) encoding genes are part of the major histocompatibility complex (MHC) on human chromosome 6. This region is one of the most polymorphic regions in the human genome. Prior knowledge of HLA allelic polymorphisms is clinically important for matching donor and recipient during organ/tissue transplantation. HLA allelic information is also useful in predicting immune responses to various infectious diseases, genetic disorders and autoimmune conditions. India harbors over a billion people and its population is untapped for HLA allelic diversity. In this study, we explored and compared three HLA typing methods for South Indian population, using Sequence-Specific Primers (SSP), NGS (Roche/454) and single- molecule sequencing (PacBio RS II) platforms. Over 1020 DNA samples were typed at low resolution using SSP method to determine the major HLA alleles within the South Indian population. These studies were followed up with medium resolution HLA typing of 80 samples based on exonic sequences on the Roche/454 sequencing system and high-resolution (6-8 digit) typing of 8 samples for HLA alleles of class I genes (HLA-A, B and C) and class II genes (HLA-DRB1 and DQB1) using PacBio RS II platform. The long reads delivered by SMRT technology, covered the full-length class I and class II genes/alleles in contiguous reads including untranslated regions, exons and introns, which provided phased SNP information. We have identified three novel alleles from PacBio data that were verified by Roche 454 sequencing. This is the first case study of HLA typing using second and third generation NGS technologies for an Indian population. The PacBio platform is a promising platform for large-scale HLA typing for establishing an HLA database for the untapped ethnic populations of India.

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July 19, 2019

Development and clinical application of an integrative genomic approach to personalized cancer therapy.

Personalized therapy provides the best outcome of cancer care and its implementation in the clinic has been greatly facilitated by recent convergence of enormous progress in basic cancer research, rapid advancement of new tumor profiling technologies, and an expanding compendium of targeted cancer therapeutics.We developed a personalized cancer therapy (PCT) program in a clinical setting, using an integrative genomics approach to fully characterize the complexity of each tumor. We carried out whole exome sequencing (WES) and single-nucleotide polymorphism (SNP) microarray genotyping on DNA from tumor and patient-matched normal specimens, as well as RNA sequencing (RNA-Seq) on available frozen specimens, to identify somatic (tumor-specific) mutations, copy number alterations (CNAs), gene expression changes, gene fusions, and also germline variants. To provide high sensitivity in known cancer mutation hotspots, Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) was also employed. We integrated the resulting data with cancer knowledge bases and developed a specific workflow for each cancer type to improve interpretation of genomic data.We returned genomics findings to 46 patients and their physicians describing somatic alterations and predicting drug response, toxicity, and prognosis. Mean 17.3 cancer-relevant somatic mutations per patient were identified, 13.3-fold, 6.9-fold, and 4.7-fold more than could have been detected using CHPv2, Oncomine Cancer Panel (OCP), and FoundationOne, respectively. Our approach delineated the underlying genetic drivers at the pathway level and provided meaningful predictions of therapeutic efficacy and toxicity. Actionable alterations were found in 91 % of patients (mean 4.9 per patient, including somatic mutations, copy number alterations, gene expression alterations, and germline variants), a 7.5-fold, 2.0-fold, and 1.9-fold increase over what could have been uncovered by CHPv2, OCP, and FoundationOne, respectively. The findings altered the course of treatment in four cases. These results show that a comprehensive, integrative genomic approach as outlined above significantly enhanced genomics-based PCT strategies.

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July 19, 2019

Shifting fitness and epistatic landscapes reflect trade-offs along an evolutionary pathway.

Nature repurposes proteins via evolutionary processes. Such adaptation can come at the expense of the original protein’s function, which is a trade-off of adaptation. We sought to examine other potential adaptive trade-offs. We measured the effect on ampicillin resistance of ~12,500 unique single amino acid mutants of the TEM-1, TEM-17, TEM-19, and TEM-15 ß-lactamase alleles, which constitute an adaptive path in the evolution of cefotaxime resistance. These protein fitness landscapes were compared and used to calculate epistatic interactions between these mutations and the two mutations in the pathway (E104K and G238S). This series of protein fitness landscapes provides a systematic, quantitative description of pairwise/tertiary intragenic epistasis involving adaptive mutations. We find that the frequency of mutations exhibiting epistasis increases along the evolutionary pathway. Adaptation moves the protein to a region in the fitness landscape characterized by decreased mutational robustness and increased ruggedness, as measured by fitness effects of mutations and epistatic interactions for TEM-1’s original function. This movement to such a “fitness territory” has evolutionary consequences and is an important adaptive trade-off and cost of adaptation. Our systematic study provides detailed insight into the relationships between mutation, protein structure, protein stability, and epistasis and quantitatively depicts the different costs inherent in the evolution of new functions. Copyright © 2016 Elsevier Ltd. All rights reserved.

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July 19, 2019

Emergence of a Homo sapiens-specific gene family and chromosome 16p11.2 CNV susceptibility.

Genetic differences that specify unique aspects of human evolution have typically been identified by comparative analyses between the genomes of humans and closely related primates, including more recently the genomes of archaic hominins. Not all regions of the genome, however, are equally amenable to such study. Recurrent copy number variation (CNV) at chromosome 16p11.2 accounts for approximately 1% of cases of autism and is mediated by a complex set of segmental duplications, many of which arose recently during human evolution. Here we reconstruct the evolutionary history of the locus and identify bolA family member 2 (BOLA2) as a gene duplicated exclusively in Homo sapiens. We estimate that a 95-kilobase-pair segment containing BOLA2 duplicated across the critical region approximately 282 thousand years ago (ka), one of the latest among a series of genomic changes that dramatically restructured the locus during hominid evolution. All humans examined carried one or more copies of the duplication, which nearly fixed early in the human lineage-a pattern unlikely to have arisen so rapidly in the absence of selection (P?

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July 19, 2019

Towards precision medicine.

There is great potential for genome sequencing to enhance patient care through improved diagnostic sensitivity and more precise therapeutic targeting. To maximize this potential, genomics strategies that have been developed for genetic discovery – including DNA-sequencing technologies and analysis algorithms – need to be adapted to fit clinical needs. This will require the optimization of alignment algorithms, attention to quality-coverage metrics, tailored solutions for paralogous or low-complexity areas of the genome, and the adoption of consensus standards for variant calling and interpretation. Global sharing of this more accurate genotypic and phenotypic data will accelerate the determination of causality for novel genes or variants. Thus, a deeper understanding of disease will be realized that will allow its targeting with much greater therapeutic precision.

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