scholarly journals Differential degradation of RNA species by autophagy related pathways in plants

2019 ◽  
Author(s):  
D. Hickl ◽  
F. Drews ◽  
C. Girke ◽  
D. Zimmer ◽  
T. Mühlhaus ◽  
...  
Keyword(s):  
Rna Processing ◽  
Length Distribution ◽  
Mature Mirnas ◽  
Read Length ◽  
Selective Transport ◽  
Rna Purification ◽  
Plant Vacuole ◽  
Generation Sequencing ◽  
Differential Degradation ◽  
Control Samples

AbstractAn important function of the plant vacuole is the recycling of the delivered proteins and RNA by autophagy. We provide the first plant vacuolar small RNome by isolation of intact vacuoles from Barley and Arabidopsis, subsequent RNA purification and Next Generation Sequencing. In these vacuolar sRNomes, all types of cellular RNAs were found including those of chloroplast origin, suggesting a bulk-type of RNA transfer to, and breakdown in vacuoles. ATG5 is a major representative of autophagy genes and the vacuolar RNA composition in corresponding knockout plants differed clearly from controls as most chloroplast derived RNA species were missing. Moreover, the read length distribution of RNAs found in ATG5 mutants differed to control samples, indicating altered RNA processing. In contrast, vacuolar RNA length and composition of plants lacking the vacuolar RNase2 (rns2-2), involved in cellular RNA homeostasis, showed minor alterations, only. Our data therefore suggests that mainly autophagy components are responsible for selective transport and targeting of different RNA species into the vacuole for degradation. In addition, mature miRNAs were detected in all vacuolar preparations, however in ATG5 mutants at much lower frequency, indicating a new biological role for vacuolar miRNAs apart from becoming degraded.

scholarly journals The effect of variant interference on de novo assembly for viral deep sequencing

2019 ◽  
Author(s):  
Christina J. Castro ◽  
Rachel L. Marine ◽  
Edward Ramos ◽  
Terry Fei Fan Ng
Keyword(s):  
Deep Sequencing ◽  
De Novo ◽  
Gc Content ◽  
Read Length ◽  
Viral Genomes ◽  
Minor Variant ◽  
Main Driver ◽  
Next Generation Sequencing Ngs ◽  
Viral Sequences ◽  
Generation Sequencing

AbstractViruses have high mutation rates and generally exist as a mixture of variants in biological samples. Next-generation sequencing (NGS) approach has surpassed Sanger for generating long viral sequences, yet how variants affect NGS de novo assembly remains largely unexplored. Our results from >15,000 simulated experiments showed that presence of variants can turn an assembly of one genome into tens to thousands of contigs. This “variant interference” (VI) is highly consistent and reproducible by ten most used de novo assemblers, and occurs independent of genome length, read length, and GC content. The main driver of VI is pairwise identities between viral variants. These findings were further supported by in silico simulations, where selective removal of minor variant reads from clinical datasets allow the “rescue” of full viral genomes from fragmented contigs. These results call for careful interpretation of contigs and contig numbers from de novo assembly in viral deep sequencing.

scholarly journals Nanopore sequencing detects structural variants in cancer

2015 ◽  
Author(s):  
Alexis L. Norris ◽  
Rachael E. Workman ◽  
Yunfan Fan ◽  
James R. Eshleman ◽  
Winston Timp
Keyword(s):  
Detection Efficiency ◽  
Electrical Current ◽  
Read Length ◽  
Therapeutic Monitoring ◽  
Base Substitution ◽  
Nanopore Sequencing ◽  
Structural Variants ◽  
Large Deletions ◽  
Long Reads ◽  
Generation Sequencing

Despite advances in sequencing, structural variants (SVs) remain difficult to reliably detect due to the short read length (<300bp) of 2nd generation sequencing. Not only do the reads (or paired-end reads) need to straddle a breakpoint, but repetitive elements often lead to ambiguities in the alignment of short reads. We propose to use the long-reads (up to 20kb) possible with 3rd generation sequencing, specifically nanopore sequencing on the MinION. Nanopore sequencing relies on a similar concept to a Coulter counter, reading the DNA sequence from the change in electrical current resulting from a DNA strand being forced through a nanometer-sized pore embedded in a membrane. Though nanopore sequencing currently has a relatively high mismatch rate that precludes base substitution and small frameshift mutation detection, its accuracy is sufficient for SV detection because of its long reads. In fact, long reads in some cases may improve SV detection efficiency. We have tested nanopore sequencing to detect a series of well-characterized SVs, including large deletions, inversions, and translocations that inactivate the CDKN2A/p16 and SMAD4/DPC4 tumor suppressor genes in pancreatic cancer. Using PCR amplicon mixes, we have demonstrated that nanopore sequencing can detect large deletions, translocations and inversions at dilutions as low as 1:100, with as few as 500 reads per sample. Given the speed, small footprint, and low capital cost, nanopore sequencing could become the ideal tool for the low-level detection of cancer-associated SVs needed for molecular relapse, early detection, or therapeutic monitoring.

scholarly journals Psip1/p52 regulates distal Hoxa genes through activation of lncRNA Hottip

2016 ◽  
Author(s):  
Madapura M Pradeepa ◽  
Gillian Taylor ◽  
Graeme R Grimes ◽  
Andrew J Wood ◽  
Wendy A Bickmore
Keyword(s):  
Rna Processing ◽  
Regulation Of Gene Expression ◽  
Embryonic Stem ◽  
Biological Functions ◽  
Interacting Protein ◽  
Rna Purification ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Hoxa Genes ◽  
In Cis

AbstractLong noncoding RNAs (lncRNAs) have been implicated in various biological functions including regulation of gene expression, X-inactivation, imprinting, cell proliferation and differentiation. However, the functionality of lncRNAs is not clearly understood and conflicting conclusions have often been reached when comparing different methods to investigate them. Moreover, little is known about the upstream regulation of lncRNAs. Here we show that a transcriptional co activator – PC4 and SF2 interacting protein (Psip1)/p52, which is involved in linking transcription to RNA processing, regulates the expression of the lncRNA Hottip. Using complementary approaches – knockdown, Cas9 mediated lncRNA deletion, analysis of lncRNA binding by Chromatin isolation by RNA purification (ChIRP) - we demonstrate that Hottip binds to the 5’ Hoxa genes located in cis, which leads to their upregulation. Moreover, the synthetic activation of Hottip is sufficient to induce the expression ofpolycomb repressed Hox genes in mouse embryonic stem cells (mESCs).

scholarly journals DR2S: An Integrated Algorithm Providing Reference-Grade Haplotype Sequences from Heterozygous Samples

2020 ◽  
Author(s):  
Steffen Klasberg ◽  
Alexander H. Schmidt ◽  
Vinzenz Lange ◽  
Gerhard Schöfl
Keyword(s):  
Allelic Variation ◽  
R Package ◽  
Full Length ◽  
Reference Sequence ◽  
Read Length ◽  
Sequencing Data ◽  
High Quality ◽  
Reference Allele ◽  
Sequencing Technologies ◽  
Generation Sequencing

AbstractBackgroundHigh resolution HLA genotyping of donors and recipients is a crucially important prerequisite for haematopoetic stem-cell transplantation and relies heavily on the quality and completeness of immuno-genetic reference sequence databases of allelic variation.ResultsHere, we report on DR2S, an R package that leverages the strengths of two sequencing technologies – the accuracy of next-generation sequencing with the read length of third-generation sequencing technologies like PacBio’s SMRT sequencing or ONT’s nanopore sequencing – to reconstruct fully-phased high-quality full-length haplotype sequences. Although optimised for HLA and KIR genes, DR2S is applicable to all loci with known reference sequences provided that full-length sequencing data is available for analysis. In addition, DR2S integrates supporting tools for easy visualisation and quality control of the reconstructed haplotype to ensure suitability for submission to public allele databases.ConclusionsDR2S is a largely automated workflow designed to create high-quality fully-phased reference allele sequences for highly polymorphic gene regions such as HLA or KIR. It has been used by biologists to successfully characterise and submit more than 500 HLA alleles and more than 500 KIR alleles to the IPD-IMGT/HLA and IPD-KIR databases.

scholarly journals Detection of structural variations and fusion genes in breast cancer samples using third-generation sequencing

2021 ◽  
Author(s):  
Taobo Hu ◽  
Jingjing Li ◽  
Mengping Long ◽  
Jinbo Wu ◽  
Zhen Zhang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Patients ◽  
Genetic Alterations ◽  
Read Length ◽  
Breast Cancer Patients ◽  
Structural Variations ◽  
Transcriptomic Sequencing ◽  
Long Read ◽  
Second Generation Sequencing ◽  
Generation Sequencing

Abstract Background: Structural variations (SVs) are common genetic alterations in the human genome that could cause different phenotypes and various diseases including cancer. However, the detection of structural variations using the second-generation sequencing was limited by its short read-length which in turn restrained our understanding of structural variations. Methods: In this study, we developed a 28-gene panel for long-read sequencing and employed it to both Oxford Nanopore Technologies and Pacific Biosciences platforms. We analyzed structural variations in the 28 breast cancer-related genes through long-read genomic and transcriptomic sequencing of tumor, para-tumor and blood samples in 19 breast cancer patients. Results: Our results showed that some somatic SVs were recurring among the selected genes, though the majority of them occurred in the non-exonic region. We found evidence supporting the existence of hotspot regions for SVs, which extended our previous understanding that they exist only for single nucleotide variations. Conclusions: In conclusion, we employed long-read genomic and transcriptomic sequencing in identifying SVs from breast cancer patients and proved that this approach holds great potential in clinical application.

scholarly journals Small-scale sequencing enables quality assessment of Ribo-Seq data: an example from Arabidopsis cell culture

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Amir Mahboubi ◽  
Nicolas Delhomme ◽  
Sara Häggström ◽  
Johannes Hanson
Keyword(s):  
Data Analysis ◽  
Quality Assessment ◽  
Deep Sequencing ◽  
Translational Regulation ◽  
Length Distribution ◽  
Great Depth ◽  
Read Length ◽  
Small Scale ◽  
Library Construction ◽  
Site Distribution

Abstract Background Translation is a tightly regulated process, controlling the rate of protein synthesis in cells. Ribosome sequencing (Ribo-Seq) is a recently developed tool for studying actively translated mRNA and can thus directly address translational regulation. Ribo-Seq libraries need to be sequenced to a great depth due to high contamination by rRNA and other contaminating nucleic acid fragments. Deep sequencing is expensive, and it generates large volumes of data, making data analysis complicated and time consuming. Methods and results Here we developed a platform for Ribo-Seq library construction and data analysis to enable rapid quality assessment of Ribo-Seq libraries with the help of a small-scale sequencer. Our data show that several qualitative features of a Ribo-Seq library, such as read length distribution, P-site distribution, reading frame and triplet periodicity, can be effectively evaluated using only the data generated by a benchtop sequencer with a very limited number of reads. Conclusion Our pipeline enables rapid evaluation of Ribo-Seq libraries, opening up possibilities for optimization of Ribo-Seq library construction from difficult samples, and leading to better decision making prior to more costly deep sequencing.

A Streamlined and High-Throughput Error-Corrected Next-Generation Sequencing Method for Low Variant Allele Frequency Quantitation

2019 ◽  
Author(s):  
Page B McKinzie ◽  
Michelle E Bishop
Keyword(s):  
Next Generation Sequencing ◽  
Suppressor Gene ◽  
Tumor Formation ◽  
Read Length ◽  
Next Generation ◽  
Sequencing Method ◽  
Proportional Increase ◽  
Duplex Sequencing ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Quantifying mutant or variable allele frequencies (VAFs) of ≤10−3 using next-generation sequencing (NGS) has utility in both clinical and nonclinical settings. Two common approaches for quantifying VAFs using NGS are tagged single-strand sequencing and duplex sequencing. While duplex sequencing is reported to have sensitivity up to 10−8 VAF, it is not a quick, easy, or inexpensive method. We report a method for quantifying VAFs that are ≥10−4 that is as easy and quick for processing samples as standard sequencing kits, yet less expensive than the kits. The method was developed using PCR fragment-based VAFs of Kras codon 12 in log10 increments from 10−5 to 10−1, then applied and tested on native genomic DNA. For both sources of DNA, there is a proportional increase in the observed VAF to input VAF from 10−4 to 100% mutant samples. Variability of quantitation was evaluated within experimental replicates and shown to be consistent across sample preparations. The error at each successive base read was evaluated to determine if there is a limit of read length for quantitation of ≥10−4, and it was determined that read lengths up to 70 bases are reliable for quantitation. The method described here is adaptable to various oncogene or tumor suppressor gene targets, with the potential to implement multiplexing at the initial tagging step. While easy to perform manually, it is also suited for robotic handling and batch processing of samples, facilitating detection and quantitation of genetic carcinogenic biomarkers before tumor formation or in normal-appearing tissue.

Detecting TYMS tandem repeat polymorphism by the PSSD method based on next generation sequecing

2020 ◽  
Vol 15 ◽  
Author(s):  
Binsheng He ◽  
Jialiang Yang ◽  
Geng Tian ◽  
Pingping Bing ◽  
Jidong Lang
Keyword(s):  
Tandem Repeat ◽  
Variable Number ◽  
Read Length ◽  
Line Treatment ◽  
Next Generation ◽  
Gene Encoding ◽  
Sequencing Errors ◽  
Novel Method ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

: Thymidylate synthase (TS) is an important target for folicacid inhibitors such as pemetrexed, which has considerable effects on the first-line treatment, second-line treatment and maintenance therapy for patients with late-stage non-small cell lung cancer (NSCLC). Therefore, detecting mutations in TYMS gene encoding TS is critical in clinical applications. With the development of the next-generation sequencing (NGS) technology, the accuracy of TYMS mutation detection is getting higher and higher. However, traditional methods suffer from false-positives and false-negatives caused by factors like limited sequencing read length and sequencing errors. In this study, we have developed a novel method based on "paired seed sequence distance” (PSSD) to detect the variable number of tandem repeat (VNTR) mutation for TYMS. Our method not only improves the detection rate and accuracy of TYMS VNTR mutations, but also avoids problems caused by sequencing errors and limited sequencing length. This method provides a new solution for similar polymorphism analyses and other sequencing analyses.

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