Advances in Single Molecule, Real-Time (SMRT) Sequencing

PacBio’s single-molecule real-time (SMRT) sequencing technology offers important advantages over the short-read DNA sequencing technologies that currently dominate the market. This includes exceptionally long read lengths (20 kb or more), unparalleled consensus accuracy, and the ability to sequence native, non-amplified DNA molecules. From fungi to insects to humans, long reads are now used to create highly accurate reference genomes by de novo assembly of genomic DNA and to obtain a comprehensive view of transcriptomes through the sequencing of full-length cDNAs. Besides reducing biases, sequencing native DNA also permits the direct measurement of DNA base modifications. Therefore, SMRT sequencing has become an attractive technology in many fields, such as agriculture, basic science, and medical research. The boundaries of SMRT sequencing are continuously being pushed by developments in bioinformatics and sample preparation. This book contains a collection of articles showcasing the latest developments and the breadth of applications enabled by SMRT sequencing technology

Global and unbiased detection of splice junctions from RNA-seq data

SplitSeek can be used to detect novel splicing events in SOLiD RNA-seq data without the need for a pre-defined library

Identification of novel exons and transcribed regions by chimpanzee transcriptome sequencing

Background: We profile the chimpanzee transcriptome by using deep sequencing of cDNA from brain and liver, aiming to quantify expression of known genes and to identify novel transcribed regions. Results: Using stringent criteria for transcription, we identify 12,843 expressed genes, with a majority being found in both tissues. We further identify 9,826 novel transcribed regions that are not overlapping with annotated exons, mRNAs or ESTs. Over 80 % of the novel transcribed regions map within or in the vicinity of known genes, and by combining sequencing data with de novo splice predictions we predict several of the novel transcribed regions to be new exons or 3 ' UTRs. For approximately 350 novel transcribed regions, the corresponding DNA sequence is absent in the human reference genome. The presence of novel transcribed regions in five genes and in one intergenic region is further validated with RT-PCR. Finally, we describe and experimentally validate a putative novel multi-exon gene that belongs to the ATP-cassette transporter gene family. This gene does not appear to be functional in human since one exon is absent from the human genome. In addition to novel exons and UTRs, novel transcribed regions may also stem from different types of noncoding transcripts. We note that expressed repeats and introns from unspliced mRNAs are especially common in our data. Conclusions: Our results extend the chimpanzee gene catalogue with a large number of novel exons and 3 ' UTRs an

Combinatorial control of gene expression by the three yeast repressors Mig1, Mig2 and Mig3

<p>Abstract</p> <p>Background</p> <p>Expression of a large number of yeast genes is repressed by glucose. The zinc finger protein Mig1 is the main effector in glucose repression, but yeast also has two related proteins: Mig2 and Mig3. We have used microarrays to study global gene expression in all possible combinations of <it>mig1</it>, <it>mig2 </it>and <it>mig3 </it>deletion mutants.</p> <p>Results</p> <p>Mig1 and Mig2 repress a largely overlapping set of genes on 2% glucose. Genes that are upregulated in a <it>mig1 mig2 </it>double mutant were grouped according to the contribution of Mig2. Most of them show partially redundant repression, with Mig1 being the major repressor, but some genes show complete redundancy, and some are repressed only by Mig1. Several redundantly repressed genes are involved in phosphate metabolism. The promoters of these genes are enriched for Pho4 sites, a novel GGGAGG motif, and a variant Mig1 site which is absent from genes repressed only by Mig1. Genes repressed only by Mig1 on 2% glucose include the hexose transporter gene <it>HXT4</it>, but Mig2 contributes to <it>HXT4 </it>repression on 10% glucose. <it>HXT6 </it>is one of the few genes that are more strongly repressed by Mig2. Mig3 does not seem to overlap in function with Mig1 and Mig2. Instead, Mig3 downregulates the <it>SIR2 </it>gene encoding a histone deacetylase involved in gene silencing and the control of aging.</p> <p>Conclusion</p> <p>Mig2 fine-tunes glucose repression by targeting a subset of the Mig1-repressed genes, and by responding to higher glucose concentrations. Mig3 does not target the same genes as Mig1 and Mig2, but instead downregulates the <it>SIR2 </it>gene.</p

SNX10 gene mutation leading to osteopetrosis with dysfunctional osteoclasts

Acknowledgements We sincerely thank the patients and family members who participated in this study. We would also like to thank Stefan Esher, Umeå University, for help with genealogy, and Anna Westerlund for excellent technical assistance. This work was supported by grants from the FOU, at the Umeå university hospital, and the Medical Faculty at Umeå University. The work at University of Gothenburg was supported by grants from The Swedish Research Council, the Swedish Rheumatism Association, the Royal 80-Year Fund of King Gustav V, ALF/LUA research grant from Sahlgrenska University Hospital in Gothenburg and the Lundberg Foundation. The work at the University of Gothenburg and the University of Aberdeen was supported by Euroclast, a Marie Curie FP7-People-2013-ITN: # 607446.Peer reviewedPublisher PD

Mutation in CEP63 co-segregating with developmental dyslexia in a Swedish family

Developmental dyslexia is the most common learning disorder in children. Problems in reading and writing are likely due to a complex interaction of genetic and environmental factors, resulting in reduced power of studies of the genetic factors underlying developmental dyslexia. Our approach in the current study was to perform exome sequencing of affected and unaffected individuals within an extended pedigree with a familial form of developmental dyslexia. We identified a two-base mutation, causing a p.R229L amino acid substitution in the centrosomal protein 63 kDa (CEP63), co-segregating with developmental dyslexia in this pedigree. This mutation is novel, and predicted to be highly damaging for the function of the protein. 3D modelling suggested a distinct conformational change caused by the mutation. CEP63 is localised to the centrosome in eukaryotic cells and is required for maintaining normal centriole duplication and control of cell cycle progression. We found that a common polymorphism in the CEP63 gene had a significant association with brain white matter volume. The brain regions were partly overlapping with the previously reported region influenced by polymorphisms in the dyslexia susceptibility genes DYX1C1 and KIAA0319. We hypothesise that CEP63 is particularly important for brain development and might control the proliferation and migration of cells when those two events need to be highly coordinated.Peer reviewe

Molecular characterization of a large unselected cohort of metastatic colorectal cancers in relation to primary tumor location, rare metastatic sites and prognosis

Background: We have reported that BRAF V600E mutations and microsatellite instability-high (MSI-H) are more prevalent in a population-based cohort of metastatic colorectal cancer (mCRC) patients than has been reported from clinical trials or hospital-based patient groups. The aim was to explore if other mutations in mCRC differ in prevalence between these cohorts in relation to mismatch repair status and primary tumor location and if presence of bone or brain metastases is associated with any mutations. Material and methods: A population-based cohort of 798 mCRC patients from three regions in Scandinavia was used. Forty-four cancer related genes were investigated in a custom designed Ampliseq hotspot panel. Differences in survival were analyzed using the Kaplan–Meier estimator and the Cox regression analysis. Results: Determination of mutations was possible in 449/501 patients for 40/44 genes. Besides BRAF V600E, seen in 19% of the tumors, none of the other mutations appeared more prevalent than in trial cohorts. BRAF V600E and MSI-H, seen in 8%, were associated with poor prognosis as was right-sided primary tumor location (39%) when compared to left-sided and rectum together; however, in a multivariable regression, only the BRAF mutation retained its statistical significance. No other mutations were associated with poor prognosis. ERBB2 alterations were more common if bone metastases were present at diagnosis (17% vs. 4%, p = .011). No association was found for brain metastases. Fifty-two percent had an alteration that is treatable with an FDA-approved targeted therapy, chiefly by EGFR-inhibitor for RAS wild-type and a check-point inhibitor for MSI-H tumors. Conclusions: Right-sided tumor location, BRAF V600E mutations, but no other investigated mutation, and MSI-H are more commonly seen in an unselected cohort than is reported from clinical patient cohorts, likely because they indicate poor prognosis. Half of the patients have a tumor that is treatable with an already FDA-approved targeted drug for mCRC.publishedVersio