Sequence based typing (SBT) is considered the gold standard method for HLA typing. Current SBT methods are rather laborious and are prone to phase ambiguity problems and genotyping uncertainties. As a result, the NGS community is rapidly seeking to remedy these challenges, to produce high resolution and high throughput HLA sequencing conducive to a clinical setting. Today, second generation NGS technologies are limited in their ability to yield full length HLA sequences required for adequate phasing and identification of novel alleles. Here we present the use of single molecule real time (SMRT) sequencing as a means of determining full length/long…
Commentary from PacBio users on their applications of SMRT Sequencing, including Ulf Gyllensten (Uppsala University), Tim Smith (USDA-ARS) and Bobby Sebra (Icahn School of Medicine)
In this AGBT 2017 poster, Ulf Gyllensten from Uppsala University presents two local reference genomes generated with PacBio and Bionano Genomics data. These assemblies include structural variation and repetitive regions that have been missed with previous short-read efforts, including some new genes not annotated in the human reference genome.
This webinar highlights global initiatives currently underway to use Single Molecule, Real-Time (SMRT) Sequencing to de novo assemble genomes of individuals representing multiple ethnic populations, thereby extending the diversity of available human reference genomes. In their presentations, Tina Graves-Lindsay from Washington University and Adam Ameur from Uppsala University spoke about diploid assemblies, discovering novel sequence and improving diversity of the current human reference genome. Finally, Paul Peluso of PacBio presented data from the recent effort to sequence a Puerto Rican genome and shared a SMRT Sequencing technology roadmap showing the next several upgrades for the Sequel System.
Meiotic drive is the preferential transmission of a particular allele during sexual reproduction. The phenomenon is observed as spore killing in multiple fungi. In natural populations of Podospora anserina, seven spore killer types (Psks) have been identified through classical genetic analyses. Here we show that the Spok gene family underlies the Psks. The combination of Spok genes at different chromosomal locations defines the spore killer types and creates a killing hierarchy within a population. We identify two novel Spok homologs located within a large (74-167 kbp) region (the Spok block) that resides in different chromosomal locations in different strains. We…
Alcaligenes aquatilis strain QD168 (= CCUG 69566) is a marine hydrocarbon-degrading bacterium isolated from crude oil-polluted sediment from Quintero Bay, Central Chile. Here, we present the 4.32-Mb complete genome sequence of strain QD168, with 3,892 coding sequences, 58 tRNAs, and a 56.3% G+C content.
Members of the Mycobacterium chelonae-abscessus complex (MCAC) are close to the mycobacterial ancestor and includes both human, animal and fish pathogens. We present the genomes of 14 members of this complex: the complete genomes of Mycobacterium salmoniphilum and Mycobacterium chelonae type strains, seven M. salmoniphilum isolates, and five M. salmoniphilum-like strains including strains isolated during an outbreak in an animal facility at Uppsala University. Average nucleotide identity (ANI) analysis and core gene phylogeny revealed that the M. salmoniphilum-like strains are variants of the human pathogen Mycobacterium franklinii and phylogenetically close to Mycobacterium abscessus. Our data further suggested that M. salmoniphilum…
Background Soaking of sorghum seeds for six hours in an aqueous extract of Eclipta alba has been shown to increase the yield of sorghum in field experiments. The effect on yield is known to depend on field location and a mechanism involving pathogen suppression has been proposed. However, it has not been clear to which extent the same effect can be obtained by soaking of seeds in pure water (hydropriming). To address this question, fifty eight field tests were conducted comparing no treatment of seeds, hydropriming and treatment with plant extract. Experiments were distributed over three years in Burkina Faso…
Revertant mosaicism (RM) is a naturally occurring phenomenon where the pathogenic effect of a germline mutation is corrected by a second somatic event. Development of healthy-looking skin due to RM has been observed in patients with various inherited skin disorders, but not in connexin-related disease. We aimed to clarify the underlying molecular mechanisms of suspected RM in the skin of a patient with keratitis-ichthyosis-deafness (KID) syndrome. The patient was diagnosed with KID syndrome due to characteristic skin lesions, hearing deficiency and keratitis. Investigation of GJB2 encoding connexin (Cx) 26 revealed heterozygosity for the recurrent de novo germline mutation, c.148G?>?A, p.Asp50Asn.…
Evolution has provided environmental bacteria with a plethora of genes that give resistance to antibiotic compounds. Under anthropogenic selection pressures, some of these genes are believed to be recruited over time into pathogens by horizontal gene transfer. River sediment polluted with fluoroquinolones and other drugs discharged from bulk drug production in India constitute an environment with unprecedented, long-term antibiotic selection pressures. It is therefore plausible that previously unknown resistance genes have evolved and/or are promoted here. In order to search for novel resistance genes, we therefore analyzed such river sediments by a functional metagenomics approach. DNA fragments providing resistance to…
Rapid advances in short-read DNA sequencing technologies have revolutionized population genomic studies, but there are genomic regions where this technology reaches its limits. Limitations mostly arise due to the difficulties in assembly or alignment to genomic regions of high sequence divergence and high repeat content, which are typical characteristics for loci under strong long-term balancing selection. Studying genetic diversity at such loci therefore remains challenging. Here, we investigate the feasibility and error rates associated with targeted long-read sequencing of a locus under balancing selection. For this purpose, we generated bacterial artificial chromosomes (BACs) containing the Brassicaceae S-locus, a region under…
Anthelmintic resistance in gastrointestinal nematode (GIN) parasites of grazing ruminants is on the rise in countries across the world. Haemonchus contortus is one of most frequently encountered drug-resistant GINs in small ruminants. This blood-sucking abomasal nematode contributes to massive treatment costs and poses a serious threat to farm animal health. To prevent the establishment of resistant strains of this parasite, up-to-date molecular techniques need to be proposed which would allow for quick, cheap and accurate identification of individuals infected with resistant worms. The effort has been made in the previous decade, with the development of the pyrosequencing method to detect…
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…
De novo mutations contribute significantly to severe early-onset genetic disorders. Even if the mutation is apparently de novo, there is a recurrence risk due to parental germ line mosaicism, depending on in which gonadal generation the mutation occurred.We demonstrate the power of using SMRT sequencing and ddPCR to determine parental origin and allele frequencies of de novo mutations in germ cells in two families whom had undergone assisted reproduction.In the first family, a TCOF1 variant c.3156C>T was identified in the proband with Treacher Collins syndrome. The variant affects splicing and was determined to be of paternal origin. It was present…
The current human reference sequence (GRCh38) is a foundation for large-scale sequencing projects. However, recent studies have suggested that GRCh38 may be incomplete and give a suboptimal representation of specific population groups. Here, we performed a de novo assembly of two Swedish genomes that revealed over 10 Mb of sequences absent from the human GRCh38 reference in each individual. Around 6 Mb of these novel sequences (NS) are shared with a Chinese personal genome. The NS are highly repetitive, have an elevated GC-content, and are primarily located in centromeric or telomeric regions. Up to 1 Mb of NS can be…