Ribosomal profiling adds new coding sequences to the proteome

2015 ◽  
Vol 43 (6) ◽  
pp. 1271-1276 ◽  
Author(s):  
Muhammad Ali S. Mumtaz ◽  
Juan Pablo Couso
Keyword(s):  
Single Nucleotide ◽  
Protein Coding ◽  
Large Numbers ◽  
New Findings ◽  
Genomic Scale ◽  
Bioinformatic Approaches ◽  
And Function ◽  
Next Generation Sequencing Ngs ◽  
Small Orfs ◽  
Generation Sequencing

Next generation sequencing (NGS) has enabled an in-depth look into genes, transcripts and their translation at the genomic scale. The application of NGS sequencing of ribosome footprints (Ribo-Seq) reveals translation with single nucleotide (nt) resolution, through the deep sequencing of ribosome-bound fragments (RBFs). Some results of Ribo-Seq challenge our understanding of the protein-coding potential of the genome. Earlier bioinformatic approaches had shown the presence of hundreds of thousands of putative small ORFs (smORFs) in eukaryotic genomes, but they had been largely ignored due to their large numbers and difficulty in determining their translation and function. Ribo-Seq has revealed that hundreds of putative smORFs within previously assumed long non-coding RNAs (lncRNAs) and UTRs of canonical mRNAs are associated with ribosomes, appearing to be translated. Here we review some of the approaches used to define translation within Ribo-Seq experiments and the challenges in defining translation of these novel smORFs in lncRNAs and UTRs. We also look at some of the bioinformatic and biochemical approaches used to independently corroborate these exciting new findings and elucidate real translation events.

scholarly journals Tiled-ClickSeq for targeted sequencing of complete coronavirus genomes with simultaneous capture of RNA recombination and minority variants

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Elizabeth Jaworski ◽  
Rose M Langsjoen ◽  
Brooke Mitchell ◽  
Barbara Judy ◽  
Patrick Newman ◽  
...  
Keyword(s):  
Full Range ◽  
Virus Genome ◽  
Clinical Samples ◽  
Rna Recombination ◽  
Single Nucleotide ◽  
Reverse Transcription Reaction ◽  
Minority Variants ◽  
Next Generation Sequencing Ngs ◽  
Unique Molecular Identifier ◽  
Generation Sequencing

High-throughput genomics of SARS-CoV-2 is essential to characterize virus evolution and to identify adaptations that affect pathogenicity or transmission. While single-nucleotide variations (SNVs) are commonly considered as driving virus adaption, RNA recombination events that delete or insert nucleic acid sequences are also critical. Whole genome targeting sequencing of SARS-CoV-2 is typically achieved using pairs of primers to generate cDNA amplicons suitable for next-generation sequencing (NGS). However, paired-primer approaches impose constraints on where primers can be designed, how many amplicons are synthesized and requires multiple PCR reactions with non-overlapping primer pools. This imparts sensitivity to underlying SNVs and fails to resolve RNA recombination junctions that are not flanked by primer pairs. To address these limitations, we have designed an approach called ‘Tiled-ClickSeq’, which uses hundreds of tiled-primers spaced evenly along the virus genome in a single reverse-transcription reaction. The other end of the cDNA amplicon is generated by azido-nucleotides that stochastically terminate cDNA synthesis, removing the need for a paired-primer. A sequencing adaptor containing a Unique Molecular Identifier (UMI) is appended to the cDNA fragment using click-chemistry and a PCR reaction generates a final NGS library. Tiled-ClickSeq provides complete genome coverage, including the 5’UTR, at high depth and specificity to the virus on both Illumina and Nanopore NGS platforms. Here, we analyze multiple SARS-CoV-2 isolates and clinical samples to simultaneously characterize minority variants, sub-genomic mRNAs (sgmRNAs), structural variants (SVs) and D-RNAs. Tiled-ClickSeq therefore provides a convenient and robust platform for SARS-CoV-2 genomics that captures the full range of RNA species in a single, simple assay.

scholarly journals Repli-seq: genome-wide analysis of replication timing by next-generation sequencing

2017 ◽  
Author(s):  
Claire Marchal ◽  
Takayo Sasaki ◽  
Daniel Vera ◽  
Korey Wilson ◽  
Jiao Sima ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Replication Timing ◽  
Nucleotide Polymorphisms ◽  
Robust Methods ◽  
Next Generation ◽  
Single Nucleotide ◽  
Cellular Processes ◽  
Genome Wide ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

ABSTRACTCycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early and late replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and sub-nuclear position. Moreover, RT is regulated during development and is altered in disease. Exploring mechanisms linking RT to other cellular processes in normal and diseased cells will be facilitated by rapid and robust methods with which to measure RT genome wide. Here, we describe a rapid, robust and relatively inexpensive protocol to analyze genome-wide RT by next-generation sequencing (NGS). This protocol yields highly reproducible results across laboratories and platforms. We also provide computational pipelines for analysis, parsing phased genomes using single nucleotide polymorphisms (SNP) for analyzing RT allelic asynchrony, and for direct comparison to Repli-chip data obtained by analyzing nascent DNA by microarrays.

scholarly journals The Mitochondrial Genomes of 18 New Pleurosticti (Coleoptera: Scarabaeidae) Exhibit a Novel trnQ-NCR-trnI-trnM Gene Rearrangement and Clarify Phylogenetic Relationships of Subfamilies within Scarabaeidae

Insects ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1025
Author(s):  
Sam Pedro Galilee Ayivi ◽  
Yao Tong ◽  
Kenneth B. Storey ◽  
Dan-Na Yu ◽  
Jia-Yong Zhang
Keyword(s):  
Next Generation Sequencing ◽  
Gene Order ◽  
Phylogenetic Relationships ◽  
Gene Rearrangement ◽  
Next Generation ◽  
Protein Coding ◽  
Protein Coding Genes ◽  
Next Generation Sequencing Ngs ◽  
Mt Genome ◽  
Generation Sequencing

The availability of next-generation sequencing (NGS) in recent years has facilitated a revolution in the availability of mitochondrial (mt) genome sequences. The mt genome is a powerful tool for comparative studies and resolving the phylogenetic relationships among insect lineages. The mt genomes of phytophagous scarabs of the subfamilies Cetoniinae and Dynastinae were under-represented in GenBank. Previous research found that the subfamily Rutelinae was recovered as a paraphyletic group because the few representatives of the subfamily Dynastinae clustered into Rutelinae, but the subfamily position of Dynastinae was still unclear. In the present study, we sequenced 18 mt genomes from Dynastinae and Cetoniinae using next-generation sequencing (NGS) to re-assess the phylogenetic relationships within Scarabaeidae. All sequenced mt genomes contained 37 sets of genes (13 protein-coding genes, 22 tRNAs, and two ribosomal RNAs), with one long control region, but the gene order was not the same between Cetoniinae and Dynastinae species. All mt genomes of Dynastinae species showed the same gene rearrangement of trnQ-NCR-trnI-trnM, whereas all mt genomes of Cetoniinae species showed the ancestral insect gene order of trnI-trnQ-trnM. Phylogenetic analyses (IQ-tree and MrBayes) were conducted using 13 protein-coding genes based on nucleotide and amino acid datasets. In the ML and BI trees, we recovered the monophyly of Rutelinae, Cetoniinae, Dynastinae, and Sericinae, and the non-monophyly of Melolonthinae. Cetoniinae was shown to be a sister clade to (Dynastinae + Rutelinae).

scholarly journals A customized scaffolds approach for the detection and phasing of complex variants by next-generation sequencing

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Qiandong Zeng ◽  
Natalia T. Leach ◽  
Zhaoqing Zhou ◽  
Hui Zhu ◽  
Jean A. Smith ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Causative Mutation ◽  
Next Generation ◽  
Single Nucleotide ◽  
Highly Sensitive ◽  
Highly Sensitive Detection ◽  
Next Generation Sequencing Ngs ◽  
Reference Bias ◽  
Generation Sequencing

Abstract Next-generation sequencing (NGS) is widely used in genetic testing for the highly sensitive detection of single nucleotide changes and small insertions or deletions. However, detection and phasing of structural variants, especially in repetitive or homologous regions, can be problematic due to uneven read coverage or genome reference bias, resulting in false calls. To circumvent this challenge, a computational approach utilizing customized scaffolds as supplementary reference sequences for read alignment was developed, and its effectiveness demonstrated with two CBS gene variants: NM_000071.2:c.833T>C and NM_000071.2:c.[833T>C; 844_845ins68]. Variant c.833T>C is a known causative mutation for homocystinuria, but is not pathogenic when in cis with the insertion, c.844_845ins68, because of alternative splicing. Using simulated reads, the custom scaffolds method resolved all possible combinations with 100% accuracy and, based on > 60,000 clinical specimens, exceeded the performance of current approaches that only align reads to GRCh37/hg19 for the detection of c.833T>C alone or in cis with c.844_845ins68. Furthermore, analysis of two 1000 Genomes Project trios revealed that the c.[833T>C; 844_845ins68] complex variant had previously been undetected in these datasets, likely due to the alignment method used. This approach can be configured for existing workflows to detect other challenging and potentially underrepresented variants, thereby augmenting accurate variant calling in clinical NGS testing.

scholarly journals Clinical Integration of Next Generation Sequencing: Coverage and Reimbursement Challenges

2014 ◽  
Vol 42 (S1) ◽  
pp. 22-41 ◽  
Author(s):  
Patricia A. Deverka ◽  
Jennifer C. Dreyfus
Keyword(s):  
Next Generation Sequencing ◽  
Information Needs ◽  
Market Demand ◽  
Next Generation ◽  
Clinical Integration ◽  
Public And Private ◽  
Regulatory Decision ◽  
Large Numbers ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Clinical next generation sequencing (NGS) is a term that refers to a variety of technologies that permit rapid sequencing of large numbers of DNA segments, up to and including entire genomes. As an approach that is playing an increasingly important role in obtaining genetic information from patients, it may be viewed by public and private payers either positively, as an enabler of the promised benefits of personalized medicine, or as “the perfect storm” resulting from the confluence of high market demand, an uproven technology, and an unprepared delivery system. A number of recent studies have noted that coverage and reimbursement will be critical for clinical integration of NGS, yet the evidentiary pathway for payer decision-making is unclear. Although there are multiple reasons for this uncertain reimbursement environment, the situation stems in large part from a long-standing lack of alignment between the information needs of regulators and post-regulatory decision-makers such as payers.

scholarly journals Seqs-Extractor: Automated sequences extraction to reduce tedious manual corrections of large datasets

2017 ◽  
Author(s):  
Patrick D C Pereira ◽  
Cleyssian Dias ◽  
Mauro A D Melo ◽  
Nara G M Magalhães ◽  
Cristovam G Diniz ◽  
...  
Keyword(s):  
Large Datasets ◽  
Local Alignment ◽  
Large Numbers ◽  
Analytical Work ◽  
Search Tool ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Generation Sequencing ◽  
Manual Correction ◽  
Reducing Potential

The analysis of large numbers of sequences requires the reduction of ambiguities during the analytical work to ensure that the effort will focus only on confirmed sequences. Performing this work automatically may help to minimize potential errors associated with tedious manual correction, allowing more effective results. Basic local alignment search tool (BLAST) seems to be the most widely used sequence analysis program. It is free, but commercial parties enhanced BLAST applications and charge a fee for their uses. There are some tools of public domain that can perform the search of microsatellites in the next generation sequencing (NGS) data, as the microsatellite identification tool (MISA), which has some features to discover microsatellites in large datasets. Here, we developed a basic shell script (BASH script) to be ran under Linux environment that can be used to extract from a sequence dataset only confirmed (BLASTed) sequences from both nucleotide (BLASTN) and protein (BLASTX) databases and extract sequences that contains microsatellites using MISA tool, using a friendly interface and no fees charged. Seqs-Extractor is a helpful tool that may enhance the analysis of large datasets in BLAST+ and MISA by minimizing the time of management, reducing potential errors caused by manipulating data and no fees charged. Seqs-Extractor is available at https://github.com/patrick-douglas/Seqs-Extractor/wiki .

scholarly journals The Complete Chloroplast Genome of Critically Endangered Chimonobambusa hirtinoda (Poaceae: Chimonobambusa) and Phylogenetic Analysis

2021 ◽  
Author(s):  
yanjiang liu ◽  
Xiao Zhu ◽  
Mingli Wu ◽  
Xue Xu ◽  
Zhaoxia Dai ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Gc Content ◽  
Trna Genes ◽  
Protein Coding ◽  
Complete Chloroplast Genome ◽  
Usage Frequency ◽  
Cp Genome ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Simple Sequence

Abstract Chimonobambusa hirtinoda is a threatened species and only naturally distributed in Doupeng Mountain, Duyun, Guizhou, China. Next-generation sequencing (NGS) is used obtained the complete chloroplast (cp) genome sequence of C. hirtinoda, and then the sequence was assembled and analyze for phylogenetic and evolutionary. We also analyzed comparing the cp genome among Chimonobambusa species with previously published. The complete cp genome of C. hirtinoda has the total length of 139, 561 bp, 38.90% GC content was detected. A total of 130 genes were founded in the cp genome, including 85 protein coding genes, 37 tRNA genes, 8 rRNA. Some genes are missing and the introns occur lost in the cp genome of C. hirtinoda. A total of 48 simple sequence repeat (SSR) were detected and by measuring the codon usage frequency of amino acids, the A/U preference of the third nucleotide in the cp genome of C. hirtinoda was obtained. Furthermore, phylogenetic analysis using complete cp sequences, matk gene exhibited genetic relationship within the Chimonobambusa genus.

Molekulare Blutgruppenbestimmung

2017 ◽  
Vol 7 (02) ◽  
pp. 87-94
Author(s):  
Christoph Gassner
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Next Generation Sequencing ◽  
Blood Transfusion ◽  
International Society ◽  
In Silico ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Molekulare Analyse ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

ZusammenfassungDie erste molekulare Analyse einer menschlichen Blutgruppe erfolgte 1983 mittels eines Restriktions-Fragment-Längen-Polymorphismus (RFLP) am System Xg. Seither wurden in unzähligen Studien die molekularen Ursachen für Blutgruppen und deren Antigene erforscht,und das resultierende Wissen für eine ständige Verbesserung der entsprechenden Analysemethoden verwendet. Die Untersuchung kausaler Punktmutationen (Single Nucleotide Polymorphism, SNP) aller 36 von der International Society for Blood Transfusion (ISBT) anerkannten Blutgruppensysteme erlaubt heute eine der Serologie ebenbürtige, exakte Vorhersage der Blutgruppenantigene. In Patienten wird die molekulare Blutgruppenbestimmung bevorzugt in Form von Einzelprobenanalytik für die Diagnose von RhD-Varianten eingesetzt, um damit transfusionsrelevante Entscheidungen bezüglich RhD zu treffen und um die Rh-Prophylaxe noch zielsicherer zu steuern. An Spenderproben und im Hochdurchsatz ermöglicht die Blutgruppen-Genotypisierung die Schaffung einer ausreichenden Anzahl von Spender-Datensätzen, um immunisierte Patienten bestverträglich zu transfundieren oder deren Immunisierung bereits im Ansatz zu vermeiden. Gleichzeitig werden heutzutage an den gleichen Proben zusätzlich eine Vielzahl weiterer SNPs zur Identifikation von Spendern mit seltener Negativität für hochfrequente Antigene getestet. Derartig umfassende Spender-Datensätze werden bereits ideal genutzt für „In-silico-Kreuzproben“ eingesetzt. Next Generation Sequencing (NGS) ist auch in der Transfusionsmedizin der „neue Stern am Horizont“ und wird vermutlich innert weniger Jahre eine wichtige Rolle in der Analyse kompletter Blutgruppengenome (chronischer) Empfänger spielen. Blutgruppenbestimmung als frühestes Beispiel echter personalisierter Medizin wird in ihrer molekularen Version mit dazu beitragen, den gebührenden Platz der Transfusionsmedizin in der modernen Medizin zu behaupten.

scholarly journals Cancer Genomics and Inherited Risk

2014 ◽  
Vol 32 (7) ◽  
pp. 687-698 ◽  
Author(s):  
Zsofia K. Stadler ◽  
Kasmintan A. Schrader ◽  
Joseph Vijai ◽  
Mark E. Robson ◽  
Kenneth Offit
Keyword(s):  
Paradigm Shift ◽  
Cancer Genomics ◽  
Massively Parallel Sequencing ◽  
Hereditary Diseases ◽  
Current State ◽  
Large Numbers ◽  
Whole Exome ◽  
Common Genetic Variants ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Next-generation sequencing (NGS) has enabled whole-exome and whole-genome sequencing of tumors for causative mutations, allowing for more accurate targeting of therapies. In the process of sequencing the tumor, comparisons to the germline genome may identify variants associated with susceptibility to cancer as well as other hereditary diseases. Already, the combination of massively parallel sequencing and selective capture approaches has facilitated efficient simultaneous genetic analysis (multiplex testing) of large numbers of candidate genes. As the field of oncology incorporates NGS approaches into tumor and germline analyses, it has become clear that the ability to achieve high-throughput genotyping surpasses our current ability to interpret and appropriately apply the vast amounts of data generated from such technologies. A review of the current state of knowledge of rare and common genetic variants associated with cancer risk or treatment outcome reveals significant progress, as well as a number of challenges associated with the clinical translation of these discoveries. The combined efforts of oncologists, genetic counselors, and cancer geneticists will be required to drive the paradigm shift toward personalized or precision medicine and to ensure the incorporation of NGS technologies into the practice of preventive oncology.

scholarly journals PEMANFAATAN TEKNOLOGI SEKUENSING GENOM UNTUK MEMPERCEPAT PROGRAM PEMULIAAN TANAMAN

2016 ◽  
Vol 34 (4) ◽  
pp. 159
Author(s):  
I Made Tasma
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Coding Region ◽  
Protein Coding ◽  
Snp Chip ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Sumber daya genetik (SDG) tanaman menyediakan materi dasar untuk program pemuliaan tanaman. Namun, baru sebagian kecil (<1%) koleksi SDG yang dimanfaatkan untuk pemuliaan tanaman. Karakterisasi SDG sudah banyak dilakukan dengan menggunakan karakter morfologi, namun metode ini lambat, menyita waktu, dan memerlukan banyak tenaga. Teknologi sekuensing modern menghasilkan peta genom rujukan suatu spesies tanaman yang   dapat mempercepat karakterisasi SDG menggunakan teknik next generation sequencing (NGS). Tulisan ini mengulas pemanfaatan teknologi sekuensing genom untuk karakterisasi, proteksi, dan pemanfaatan SDG untuk mempercepat program pemuliaan tanaman. Di Indonesia, teknologi NGS telah dimanfaatkan sejak 2010 untuk resekuensing genom komoditas unggulan nasional seperti kedelai, kakao, jagung, dan cabai merah. Jutaan SNP dan Indel telah diidentifikasi pada setiap komoditas sebagai sumber daya pemuliaan yang bernilai tinggi. Sebagian kecil SNP/Indel tersebut berada pada protein coding region yang potensial untuk penemuan gen-gen unggul. Selain SNP yang diidentifikasi pada semua genotipe, ditemukan SNP pada genotipe tertentu (SNP unik). Koleksi SNP dalam jumlah besar ini digunakan untuk mensintesis SNP chip untuk genotyping SDG secara cepat dan komprehensif. Didukung data fenotipe, SNP chip bermanfaat untuk melabel gen-gen unggul. Marka SNP yang berpautan dengan karakter unggul digunakan untuk menyeleksi individu pembawa karakter unggul tersebut. Dengan teknologi NGS, perakitan VUB tanaman dapat dilakukan lebih cepat, akurat, dan efisien. Dengan demikian, teknologi NGS dapat memfasilitasi karakterisasi dan pemanfaatan SDG untuk mem-percepat program pemuliaan tanaman.

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