Screening and Identification of PLK1-Polo Box Binding Peptides by High-Throughput Sequencing of Phage-Selected Libraries

2019 ◽  
Vol 26 (8) ◽  
pp. 620-633 ◽  
Author(s):  
Nousheen Bibi ◽  
Hafsa Niaz ◽  
Ted Hupp ◽  
Mohammad Amjad Kamal ◽  
Sajid Rashid
Keyword(s):  
Next Generation Sequencing ◽  
Phage Display ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
B Lymphocyte ◽  
Fluorescent Protein ◽  
Next Generation ◽  
Peptide Phage Display ◽  
Binding Peptides ◽  
Generation Sequencing

Background: Human proteome contains a plethora of short linear peptide motifs that is crucial for signaling and other cellular processes. These motifs are difficult to identify due to lack of systematic approach for their detection. Objective: Here we demonstrate the use of peptide phage display in combination with high throughput next generation sequencing to identify enriched peptide sequences through biopanning process against polo box domain (PBD) of mitotic polo like kinase 1 (Plk1). Methods: Purified recombinant Plk1 and two unrelated controls namely B-lymphocyte antigen (CD20) and fluorescent protein (mCherry) were subjected to peptide phage display analysis. Bacterially-propagated phage DNA was amplified by PCR using triplet bar coded primers to tag the pool from each amplicon. Results: Proteomic peptide phage display along with next generation sequencing and Bioinformatics analysis demonstrated several known and putative novel interactions which were potentially related to Plk1-PBD. With our strategy, we were able to identify and characterize several Plk1-PBD binding peptides, as well as define more precisely, consensus sequences. Conclusion: We believe that this information could provide valuable tools for exploring novel interaction involved in Plk1 signaling as well as to choose peptides for Plk1 specific drug development.

scholarly journals An integrated pipeline for next generation sequencing and annotation of the complete mitochondrial genome of the giant intestinal fluke, Fasciolopsis buski (Lankester,1857) Looss, 1899

2013 ◽  
Author(s):  
Devendra Kumar Biswal ◽  
Sudeep Ghatani ◽  
Jollin A Shylla ◽  
Ranjana Sahu ◽  
Nandita Mullapudi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Mitochondrial Genome ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Next Generation ◽  
Genome Sequences ◽  
Fasciolopsis Buski ◽  
Mt Genome ◽  
Generation Sequencing ◽  
Control Regions

Helminths include both parasitic nematodes (roundworms) and platyhelminths (trematode and cestode flatworms) that are abundant, and are of clinical importance. The genetic characterization of parasitic flatworms using advanced molecular tools is central to the diagnosis and control of infections. Although the nuclear genome houses suitable genetic markers (e.g., in ribosomal (r) DNA) for species identification and molecular characterization, the mitochondrial (mt) genome consistently provides a rich source of novel markers for informative systematics and epidemiological studies. In the last decade, there have been some important advances in mtDNA genomics of helminths, especially lung flukes, liver flukes and intestinal flukes. Fasciolopsis buski, often called the giant intestinal fluke, is one of the largest digenean trematodes infecting humans and found primarily in Asia, in particular the Indian subcontinent. Next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation within a short span of time. Herein, we describe a high-throughput sequencing and bioinformatics pipeline for mt genomics for F. buski that emphasizes the utility of short read NGS platforms such as Ion Torrent and Illumina in successfully sequencing and assembling the mt genome using innovative approaches for PCR primer design as well as assembly. We took advantage of our NGS whole genome sequence data (unpublished so far) for F. buski and its comparison with available data for the Fasciola hepatica mtDNA as the reference genome for design of precise and specific primers for amplification of mt genome sequences from F. buski. A long-range PCR was carried out to create a NGS library enriched in mt DNA sequences. Two different NGS platforms were employed for complete sequencing, assembly and annotation of the F. buski mt genome. The complete mt genome sequences of the intestinal fluke comprise 14,118 bp and is thus the shortest trematode mitochondrial genome sequenced to date. The noncoding control regions are separated into two parts by the tRNA-Gly gene and donot contain either tandem repeats or secondary structures, which are typical for trematode control regions. The gene content and arrangement are identical to that of F. hepatica. The F. buski mtDNA genome has a close resemblance with F. hepatica and has a similar gene order tallying with that of other trematodes. The mtDNA for the intestinal fluke is reported herein for the first time by our group that would help invesigate Fasciolidae taxonomy and systematics with the aid of mtDNA NGS data. More so, it would serve as a resource for comparative mitochondrial genomics and systematic studies of trematode parasites.

scholarly journals An integrated pipeline for next generation sequencing and annotation of the complete mitochondrial genome of the giant intestinal fluke, Fasciolopsis buski (Lankester,1857) Looss, 1899

2013 ◽  
Author(s):  
Devendra Kumar Biswal ◽  
Sudeep Ghatani ◽  
Jollin A Shylla ◽  
Ranjana Sahu ◽  
Nandita Mullapudi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Mitochondrial Genome ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Next Generation ◽  
Genome Sequences ◽  
Fasciolopsis Buski ◽  
Mt Genome ◽  
Generation Sequencing ◽  
Control Regions

Helminths include both parasitic nematodes (roundworms) and platyhelminths (trematode and cestode flatworms) that are abundant, and are of clinical importance. The genetic characterization of parasitic flatworms using advanced molecular tools is central to the diagnosis and control of infections. Although the nuclear genome houses suitable genetic markers (e.g., in ribosomal (r) DNA) for species identification and molecular characterization, the mitochondrial (mt) genome consistently provides a rich source of novel markers for informative systematics and epidemiological studies. In the last decade, there have been some important advances in mtDNA genomics of helminths, especially lung flukes, liver flukes and intestinal flukes. Fasciolopsis buski, often called the giant intestinal fluke, is one of the largest digenean trematodes infecting humans and found primarily in Asia, in particular the Indian subcontinent. Next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation within a short span of time. Herein, we describe a high-throughput sequencing and bioinformatics pipeline for mt genomics for F. buski that emphasizes the utility of short read NGS platforms such as Ion Torrent and Illumina in successfully sequencing and assembling the mt genome using innovative approaches for PCR primer design as well as assembly. We took advantage of our NGS whole genome sequence data (unpublished so far) for F. buski and its comparison with available data for the Fasciola hepatica mtDNA as the reference genome for design of precise and specific primers for amplification of mt genome sequences from F. buski. A long-range PCR was carried out to create a NGS library enriched in mt DNA sequences. Two different NGS platforms were employed for complete sequencing, assembly and annotation of the F. buski mt genome. The complete mt genome sequences of the intestinal fluke comprise 14,118 bp and is thus the shortest trematode mitochondrial genome sequenced to date. The noncoding control regions are separated into two parts by the tRNA-Gly gene and donot contain either tandem repeats or secondary structures, which are typical for trematode control regions. The gene content and arrangement are identical to that of F. hepatica. The F. buski mtDNA genome has a close resemblance with F. hepatica and has a similar gene order tallying with that of other trematodes. The mtDNA for the intestinal fluke is reported herein for the first time by our group that would help invesigate Fasciolidae taxonomy and systematics with the aid of mtDNA NGS data. More so, it would serve as a resource for comparative mitochondrial genomics and systematic studies of trematode parasites.

scholarly journals Assessment of Bioleaching Microbial Community Structure and Function Based on Next-Generation Sequencing Technologies

Minerals ◽  
2018 ◽  
Vol 8 (12) ◽  
pp. 596 ◽  
Author(s):  
Shuang Zhou ◽  
Min Gan ◽  
Jianyu Zhu ◽  
Xinxing Liu ◽  
Guanzhou Qiu
Keyword(s):  
Community Structure ◽  
Next Generation Sequencing ◽  
Microbial Ecology ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Structure And Function ◽  
Next Generation ◽  
Sequencing Technologies ◽  
And Function ◽  
Generation Sequencing

It is widely known that bioleaching microorganisms have to cope with the complex extreme environment in which microbial ecology relating to community structure and function varies across environmental types. However, analyses of microbial ecology of bioleaching bacteria is still a challenge. To address this challenge, numerous technologies have been developed. In recent years, high-throughput sequencing technologies enabling comprehensive sequencing analysis of cellular RNA and DNA within the reach of most laboratories have been added to the toolbox of microbial ecology. The next-generation sequencing technology allowing processing DNA sequences can produce available draft genomic sequences of more bioleaching bacteria, which provides the opportunity to predict models of genetic and metabolic potential of bioleaching bacteria and ultimately deepens our understanding of bioleaching microorganism. High-throughput sequencing that focuses on targeted phylogenetic marker 16S rRNA has been effectively applied to characterize the community diversity in an ore leaching environment. RNA-seq, another application of high-throughput sequencing to profile RNA, can be for both mapping and quantifying transcriptome and has demonstrated a high efficiency in quantifying the changing expression level of each transcript under different conditions. It has been demonstrated as a powerful tool for dissecting the relationship between genotype and phenotype, leading to interpreting functional elements of the genome and revealing molecular mechanisms of adaption. This review aims to describe the high-throughput sequencing approach for bioleaching environmental microorganisms, particularly focusing on its application associated with challenges.

scholarly journals A REVIEW ON NEXT-GENERATION SEQUENCING SAMPLE PREPARATION

2017 ◽  
Vol 1 (1) ◽  
Author(s):  
A. K. Gupta
Keyword(s):  
Next Generation Sequencing ◽  
Sample Preparation ◽  
Genome Sequencing ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Test Procedure ◽  
Next Generation ◽  
Specialized Exhibition ◽  
Generation Sequencing

To stay aware of interest for sample arrangement, numerous high-throughput sequencing labs have computerized specimen arrangement strategies. The point when selecting a robotization stage, clients might as well recognize the specialized exhibition of the fluid handler and its demonstrated exhibition for NGS test procedure. NGS test arrangement stages may as well offer clients a critical decrease in involved time and the adaptability to run numerous methodologies. In light of the fact that the sample readiness protocol for NGS are quickly developing, a perfect robotization framework ought to be good with reagents for different requirements on advancing sequencing stages.Key words: NGS, genome sequencing, BenchCel Workstation, Benchbot Robot, Bravo platform, automation protocols

scholarly journals Automated genotyping of microsatellite loci from feces with high throughput sequences

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258906
Author(s):  
Isabel Salado ◽  
Alberto Fernández-Gil ◽  
Carles Vilà ◽  
Jennifer A. Leonard
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Microsatellite Loci ◽  
High Throughput Sequencing ◽  
Individual Identification ◽  
Next Generation Sequencing Data ◽  
Next Generation ◽  
Sequencing Data ◽  
Software Packages ◽  
Generation Sequencing

Ecological and conservation genetic studies often use noninvasive sampling, especially with elusive or endangered species. Because microsatellites are generally short in length, they can be amplified from low quality samples such as feces. Microsatellites are highly polymorphic so few markers are enough for reliable individual identification, kinship determination, or population characterization. However, the genotyping process from feces is expensive and time consuming. Given next-generation sequencing (NGS) and recent software developments, automated microsatellite genotyping from NGS data may now be possible. These software packages infer the genotypes directly from sequence reads, increasing throughput. Here we evaluate the performance of four software packages to genotype microsatellite loci from Iberian wolf (Canis lupus) feces using NGS. We initially combined 46 markers in a single multiplex reaction for the first time, of which 19 were included in the final analyses. Megasat was the software that provided genotypes with fewer errors. Coverage over 100X provided little additional information, but a relatively high number of PCR replicates were necessary to obtain a high quality genotype from highly unoptimized, multiplexed reactions (10 replicates for 18 of the 19 loci analyzed here). This could be reduced through optimization. The use of new bioinformatic tools and next-generation sequencing data to genotype these highly informative markers may increase throughput at a reasonable cost and with a smaller amount of laboratory work. Thus, high throughput sequencing approaches could facilitate the use of microsatellites with fecal DNA to address ecological and conservation questions.

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