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Tuesday, April 21, 2020

FLAM-seq: full-length mRNA sequencing reveals principles of poly(A) tail length control.

Although messenger RNAs are key molecules for understanding life, until now, no method has existed to determine the full-length sequence of endogenous mRNAs including their poly(A) tails. Moreover, although non-A nucleotides can be incorporated in poly(A) tails, there also exists no method to accurately sequence them. Here, we present full-length poly(A) and mRNA sequencing (FLAM-seq), a rapid and simple method for high-quality sequencing of entire mRNAs. We report a complementary DNA library preparation method coupled to single-molecule sequencing to perform FLAM-seq. Using human cell lines, brain organoids and Caenorhabditis elegans we show that FLAM-seq delivers high-quality full-length mRNA sequences for…

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Tuesday, April 21, 2020

Genome-wide de novo L1 Retrotransposition Connects Endonuclease Activity with Replication.

L1 retrotransposon-derived sequences comprise approximately 17% of the human genome. Darwinian selective pressures alter L1 genomic distributions during evolution, confounding the ability to determine initial L1 integration preferences. Here, we generated high-confidence datasets of greater than 88,000 engineered L1 insertions in human cell lines that act as proxies for cells that accommodate retrotransposition in vivo. Comparing these insertions to a null model, in which L1 endonuclease activity is the sole determinant dictating L1 integration preferences, demonstrated that L1 insertions are not significantly enriched in genes, transcribed regions, or open chromatin. By comparison, we provide compelling evidence that the L1 endonuclease disproportionately cleaves…

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Tuesday, April 21, 2020

Double PIK3CA mutations in cis increase oncogenicity and sensitivity to PI3Ka inhibitors.

Activating mutations in PIK3CA are frequent in human breast cancer, and phosphoinositide 3-kinase alpha (PI3Ka) inhibitors have been approved for therapy. To characterize determinants of sensitivity to these agents, we analyzed PIK3CA-mutant cancer genomes and observed the presence of multiple PIK3CA mutations in 12 to 15% of breast cancers and other tumor types, most of which (95%) are double mutations. Double PIK3CA mutations are in cis on the same allele and result in increased PI3K activity, enhanced downstream signaling, increased cell proliferation, and tumor growth. The biochemical mechanisms of dual mutations include increased disruption of p110a binding to the inhibitory…

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Tuesday, April 21, 2020

Extreme resistance to Potato virus Y in potato carrying the Rysto gene is mediated by a TIR-NLR immune receptor.

Potato virus Y (PVY) is a major potato (Solanum tuberosum L.) pathogen that causes severe annual crop losses worth billions of dollars worldwide. PVY is transmitted by aphids, and successful control of virus transmission requires the extensive use of environmentally damaging insecticides to reduce vector populations. Rysto , from the wild relative S. stoloniferum, confers extreme resistance (ER) to PVY and related viruses and is a valuable trait that is widely employed in potato resistance breeding programmes. Rysto was previously mapped to a region of potato chromosome XII, but the specific gene has not been identified to date. In this…

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Tuesday, April 21, 2020

Precise therapeutic gene correction by a simple nuclease-induced double-stranded break.

Current programmable nuclease-based methods (for example, CRISPR-Cas9) for the precise correction of a disease-causing genetic mutation harness the homology-directed repair pathway. However, this repair process requires the co-delivery of an exogenous DNA donor to recode the sequence and can be inefficient in many cell types. Here we show that disease-causing frameshift mutations that result from microduplications can be efficiently reverted to the wild-type sequence simply by generating a DNA double-stranded break near the centre of the duplication. We demonstrate this in patient-derived cell lines for two diseases: limb-girdle muscular dystrophy type 2G (LGMD2G)1 and Hermansky-Pudlak syndrome type 1 (HPS1)2. Clonal…

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Tuesday, April 21, 2020

Immunogenetic factors driving formation of ultralong VH CDR3 in Bos taurus antibodies.

The antibody repertoire of Bos taurus is characterized by a subset of variable heavy (VH) chain regions with ultralong third complementarity determining regions (CDR3) which, compared to other species, can provide a potent response to challenging antigens like HIV env. These unusual CDR3 can range to over seventy highly diverse amino acids in length and form unique ß-ribbon ‘stalk’ and disulfide bonded ‘knob’ structures, far from the typical antigen binding site. The genetic components and processes for forming these unusual cattle antibody VH CDR3 are not well understood. Here we analyze sequences of Bos taurus antibody VH domains and find…

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Tuesday, April 21, 2020

Full-length mRNA sequencing in Saccharina japonica and identification of carbonic anhydrase genes

The carbonic anhydrases (CAs) are a group of enzymes that play an important role in the absorption and transportation of CO2 in Saccharina japonica. They are encoded by a superfamily of genes with seven subtypes that are unrelated in sequence but share conserved function in catalyzing the reversible conversion of CO2 and HCO3-. Here we have characterized the CA members in the transcriptome of S. japonica using Single-molecule real-time (SMRT) sequencing technology. Approximately 9830.4 megabases from 5,028,003 quality subreads were generated, and they were assembled into 326,512 full-length non-chimeric (FLNC) reads, with an average flnc read length of 2181 bp.…

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Tuesday, April 21, 2020

Newly designed 16S rRNA metabarcoding primers amplify diverse and novel archaeal taxa from the environment.

High-throughput studies of microbial communities suggest that Archaea are a widespread component of microbial diversity in various ecosystems. However, proper quantification of archaeal diversity and community ecology remains limited, as sequence coverage of Archaea is usually low owing to the inability of available prokaryotic primers to efficiently amplify archaeal compared to bacterial rRNA genes. To improve identification and quantification of Archaea, we designed and validated the utility of several primer pairs to efficiently amplify archaeal 16S rRNA genes based on up-to-date reference genes. We demonstrate that several of these primer pairs amplify phylogenetically diverse Archaea with high sequencing coverage, outperforming…

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Tuesday, April 21, 2020

Potential of TLR-gene diversity in Czech indigenous cattle for resistance breeding as revealed by hybrid sequencing

A production herd of Czech Simmental cattle (Czech Red Pied, CRP), the conserved subpopulation of this breed, and the ancient local breed Czech Red cattle (CR) were screened for diversity in the antibacterial toll-like receptors (TLRs), which are members of the innate immune system. Polymerase chain reaction (PCR) amplicons of TLR1, TLR2, TLR4, TLR5, and TLR6 from pooled DNA samples were sequenced with PacBio technology, with 3–5×?coverage per gene per animal. To increase the reliability of variant detection, the gDNA pools were sequenced in parallel with the Illumina X-ten platform at low coverage (60× per gene). The diversity in conserved…

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Tuesday, April 21, 2020

Varieties of immunity activities and gut contents in tilapia with seasonal changes.

We performed 16S rDNA sequencing of tilapia fecal samples to analyze changes in tilapia gut contents after cultivation of the fish in the presence of sandwich-like floating beds of Chinese medicinal herbs (5 and 10% planting-areas; 5% Polygonum cuspidatum). The interactive effects between water quality and blood and hepatic pro- and anti-inflammatory concentrations were also assessed. Our results showed that the water quality (i.e., NO3-N, NO2-N, TP removal rates) improved, and the abundance of Chloroflexi and Cyanobacteria increased. The abundance of Bacteroidetes, Verrucomicrobia, Saccharibacteria, and Actinobacteria showed both significant seasonal decreases and increases in the presence of P. cuspidatum (increases…

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Tuesday, April 21, 2020

Microsatellite marker set for genetic diversity assessment of primitive Chitala chitala (Hamilton, 1822) derived through SMRT sequencing technology.

In present study, single molecule-real time sequencing technology was used to obtain a validated set of microsatellite markers for application in population genetics of the primitive fish, Chitala chitala. Assembly of circular consensus sequencing reads resulted into 1164 sequences which contained 2005 repetitive motifs. A total of 100 sequences were used for primer designing and amplification yielded a set of 28 validated polymorphic markers. These loci were used to genotype n?=?72 samples from three distant riverine populations of India, namely Son, Satluj and Brahmaputra, for determining intraspecific genetic variation. The microsatellite loci exhibited high level of polymorphism with PIC values…

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Tuesday, April 21, 2020

Genome-wide analysis of methyl jasmonate-regulated isoform expression in the medicinal plant Andrographis paniculata

Alternative splicing can increase the complexity of the transcriptome and proteome. The most common mechanism of alternative splicing in plants is intron retention (IR), and the expression levels of IR isoforms can be differentially regulated when facing abiotic stress. The full-length transcriptome of the medicinal plant Andrographis paniculata was sequenced using both Illumina- and SMRT-based RNA-seq and a total of 4846 IR isoforms were identified. The expression levels of 310/296 IR isoforms were up-regulated, and 629/659 IR isoforms were down-regulated at 24?h/48?h after methyl jasmonate (MeJA) treatment, respectively. In the (E,E,E)-geranylgeranyl diphosphate (GGPP) biosynthesis pathway which contributes to the andrographolide…

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Tuesday, April 21, 2020

Determining stoichiometry and kinetics of two thermophilic nitrifying communities as a crucial step in the development of thermophilic nitrogen removal.

Nitrification and denitrification, the key biological processes for thermophilic nitrogen removal, have separately been established in bioreactors at 50?°C. A well-characterized set of kinetic parameters is essential to integrate these processes while safeguarding the autotrophs performing nitrification. Knowledge on thermophilic nitrifying kinetics is restricted to isolated or highly enriched batch cultures, which do not represent bioreactor conditions. This study characterized the stoichiometry and kinetics of two thermophilic (50?°C) nitrifying communities. The most abundant ammonia oxidizing archaea (AOA) were related to the Nitrososphaera genus, clustering relatively far from known species Nitrososphaera gargensis (95.5% 16S rRNA gene sequence identity). The most abundant…

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Tuesday, April 21, 2020

A Rigorous Interlaboratory Examination of the Need to Confirm Next-Generation Sequencing-Detected Variants with an Orthogonal Method in Clinical Genetic Testing.

Orthogonal confirmation of next-generation sequencing (NGS)-detected germline variants is standard practice, although published studies have suggested that confirmation of the highest-quality calls may not always be necessary. The key question is how laboratories can establish criteria that consistently identify those NGS calls that require confirmation. Most prior studies addressing this question have had limitations: they have been generally of small scale, omitted statistical justification, and explored limited aspects of underlying data. The rigorous definition of criteria that separate high-accuracy NGS calls from those that may or may not be true remains a crucial issue. We analyzed five reference samples and…

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Tuesday, April 21, 2020

PacBio full-length cDNA sequencing integrated with RNA-seq reads drastically improves the discovery of splicing transcripts in rice.

In eukaryotes, alternative splicing (AS) greatly expands the diversity of transcripts. However, it is challenging to accurately determine full-length splicing isoforms. Recently, more studies have taken advantage of Pacific Bioscience (PacBio) long-read sequencing to identify full-length transcripts. Nevertheless, the high error rate of PacBio reads seriously offsets the advantages of long reads, especially for accurately identifying splicing junctions. To best capitalize on the features of long reads, we used Illumina RNA-seq reads to improve PacBio circular consensus sequence (CCS) quality and to validate splicing patterns in the rice transcriptome. We evaluated the impact of CCS accuracy on the number and…

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