HITAC-seq enables high-throughput cost-effective sequencing of plasmids and DNA fragments with identity

DNA sequencing is vital for many aspects of biological research and diagnostics. Despite the development of second and third generation sequencing technologies, Sanger sequencing has long been the only choice when required to precisely track each sequenced plasmids or DNA fragments. Here, we report a complete set of novel barcoding and assembling system, Highly-parallel Indexed Tagmentation-reads Assembled Consensus sequencing(HITAC-seq), that could massively sequence and track the identities of each individual sequencing sample. With the cost of much less than that of single read of Sanger sequencing,HITAC-seq can generate high-quality contiguous sequences of up to 10 kilobases or longer. The capability of HITAC-seq was confirmed through large-scale sequencing of thousands of plasmid clones and hundreds of amplicon fragments using approximately 100 pg of input DNAs. Due to its long synthetic length, HITACseq was effective in detecting relatively large structural variations, as demonstrated by the identification of a～1.3 kb Copia retrotransposon insertion in the upstream of a likely maize domestication gene. Besides being a practical alternative to traditional Sanger sequencing, HITAC-seq is suitable for many highthroughput sequencing and genotyping applications.     

Plant-herbivorous insect networks: who is eating what revealed by long barcodes using high-throughput sequencing and Trinity assembly

Interactions between plants and insects are among the most important life functions for all organism at a particular natural community.Usually a large number of samples are required to identify insect diets in food web studies.Previously,Sanger sequencing and next generation sequencing(NGS)with short DNA barcodes were used,resulting in low species-level identification;meanwhile the costs of Sanger sequencing are expensive for metabarcoding together with more samples.Here,we present a fast and effective sequencing strategy to identify larvae of Lepidoptera and their diets at the same time without increasing the cost on Illumina platform in a single HiSeq run,with long-multiplexmetabarcoding(COI for insects,rbcL,matK,ITS and trnL for plants)obtained by Trinity assembly(SHMMT).Meanwhile,Sanger sequencing(for single individuals)and NGS(for polyphagous)were used to verify the reliability of the SHMMT approach.Furthermore,we show that SHMMT approach is fast and reliable,with most high-quality sequences of five DNA barcodes of 63 larvae individuals(54 species)recovered(full length of 100%of the COI gene and 98.3%of plant DNA barcodes)using Trinity assembly(up-sized to 1015 bp).For larvae diets identification,95%are reliable;the other 5%failed because their guts were empty.The diets identified by SHMMT approach are 100%consistent with the host plants that the larvae were feeding on during our collection.Our study demonstrates that SHMMT approach is reliable and cost-effective for insect-plants network studies.This will facilitate insect-host plant studies that generally contain a huge number of samples.     

TOTAL SYNTHESIS OF CRYSTALLINE BOVINE INSULIN

<正>We should like to communicate,as a result of our combined efforts for the past five years,this preliminary report on the total synthesis of crystalline bovine insulin,the structure of which was first elucidated by Sanger and coworkers[1]ten years ago.A number of papers on the synthetic work concerning insulin has been published during the past two years.Katsoyannis and coworkers[2,3].synthesized both the A and B chains of sheep insulin and,by combining them together according to the procedure of Dixon and Wardlow[4],observed only traces of insulin activity.Zahn,Meienhofer and co-     

Functional characterization of novel NPRL3 mutations identified in three families with focal epilepsy

Focal epilepsy accounts for 60% of all forms of epilepsy, but the pathogenic mechanism is not well understood. In this study,three novel mutations in NPRL3(nitrogen permease regulator-like 3), c.937＿945del, c.1514dup C and 6,706-bp genomic DNA(g DNA) deletion, were identified in three families with focal epilepsy by linkage analysis, whole exome sequencing(WES) and Sanger sequencing. NPRL3 protein is a component of the GATOR1 complex, a major inhibitor of m TOR signaling. These mutations led to ...     

COL4A3 mutations cause focal segmenta glomerulosclerosis

Focal segmental glomerulosclerosis （FSGS） is a histologically identifiable gtomerular injury often leading to proteinuria and renal failure. To identify its causal genes, whole-exome sequencing and Sanger sequencing were performed on a large Chinese cohort that comprised 40 FSGS families, 50 sporadic FSGS patients, 9 independent autosomal recessive Atport＇s syndrome （ARAS） patients, and 190 ethnically matched healthy controls. Patients with extrarenal manifestations, indicating systemic diseases or other known hereditary renal diseases, were excluded. Heterozygous COL4A3 mutations were identified in five （12.5%） FSGS families and one （2%） sporadic FSGS patient. All identified mutations disrupted highly conserved protein sequences and none of them was found in either public databases or the 190 healthy controls. Of the FSGS patients with heterozygous COL4A3 mutations, segmental thinning of the glomerular base membrane （GBM） was only detected in the patient with electronic microscopy examination results available. Five ARAS patients （55.6%） had homozygous or compound-heterozygous mutations in COL4.43 or COL4A4. Serious changes in the G BM, hearing loss, and ocular abnormalities were found in 100%, 80%, and 40% of the ARAS patients, respectively. Overall, a new sub- group of FSGS patients resulting from heterozygous C01.4A3 mutations was identified. The mutations are relatively frequent in famiUes diagnosed with inherited forms of FSGS. Thus, we suggest screening for C01.4A3 mutations in familial FSGS patients.     

The failed invasion of Harmonia axyridis in the Azores,Portugal: Climatic restriction or wrong population origin?

We tested two questions:(i)whether the climatic conditions of the Azorean Islands in Portugal may have restricted the invasion of Harmonia axyridis across this archipelago and(ii)determine what population of this species could have a higher probability of invading the islands.We used MaxEnt to project the climate requirements of different H.axyridis populations from three regions of the world,and the potential global niche of the species in the Azorean islands.Then we assessed the suitability of the islands for each of the three H.axyridis populations and global potential niche through histograms analysis,Principal Component Analysis(PCA)of climate variables,and a variable-by-variable assessment of the suitability response curves compared with the climatic conditions of the Azores.Climatic conditions of the Azores are less suitable for the U.S.and native Asian populations of H.axyridis,and more suitable for European populations and the global potential niche.The PCA showed that the climatic conditions of the islands differed from the climatic requirements of H.axyridis.This difference is mainly explained by precipitation of the wettest month,isothermality,and the minimum temperature of the coldest month.We concluded that the climatic conditions of the Azores could have influenced the establishment and spread of H.axyridis on these islands from Europe.Our results showed that abiotic resistance represented by the climate of the potentially colonizable zones could hinder the establishment of invasive insects,but it could vary depending of the origin of the colonizing population.     

Hydrodynamic Analysis of C-start in Crucian Carp

The kinematics of turning maneuvers of startled Crucian Carp (Carassius auratus) are presented. All escape response observed are C-type fast-starts. The position of the center of mass and the me,merit of inertia of the fish are calculated. The results show that the position of the center of mass is always at 35% of the length of the fish from the head and the position of the center of mass and rroment of inertia can be considered unchanged during C-start of Crucian Carp. Hydro-dynamic analysis of the C-start is given based on the kinematics data from our experiments. The C-start consists of three stages. In stage 1, the tail fin of fish rapidly flaps in one direction, and a large moment acts on the fish‘s body, which rotates around the center of mass with an angular acceleration. In stage 2, the tail fin flaps more slowly in the opposite direction at slower speed, the fish‘s body rotates around the center of mass with angular deceleration and the center of mass of the fish moves along an are. In stage 3, the moment approximately equals zero, the fish‘s body stops rotating and the center of mass the moves along a straight line.