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The Journal of Applied Laboratory... Jan 2024Pharmacogenetics or pharmacogenomics (PGx) is the study of the role of inherited or acquired sequence change in drug response. With the rapid evolution of molecular...
BACKGROUND
Pharmacogenetics or pharmacogenomics (PGx) is the study of the role of inherited or acquired sequence change in drug response. With the rapid evolution of molecular techniques, bioinformatic tools, and increased throughput of functional genomic studies, the discovery of PGx associations and clinical implementation of PGx test results have now moved beyond a handful variants in single pharmacogenes and multi-gene panels that interrogate a few pharmacogenes to whole-exome and whole-genome scales. Although some laboratories have adopted next-generation sequencing (NGS) as a testing platform for PGx and other molecular tests, most clinical laboratories that offer PGx tests still use targeted genotyping approaches.
CONTENT
This article discusses primarily the technical considerations for clinical laboratories to develop NGS-based PGx tests including whole-genome and whole-exome sequencing analyses and highlights the challenges and opportunities in test design, content selection, bioinformatic pipeline for PGx allele and diplotype assignment, rare variant classification, reporting, and briefly touches a few additional areas that are important for successful clinical implementation of PGx results.
SUMMARY
The accelerated speed of technology development associated with continuous cost reduction and enhanced ability to interrogate complex genome regions makes it inevitable for most, if not all, clinical laboratories to transition PGx testing to an NGS-based platform in the near future. It is important for laboratories and relevant professional societies to recognize both the potential and limitations of NGS-based PGx profiling, and to work together to develop a standard and consistent practice to maximize the variant or allele detection rate and utility of PGx testing.
Topics: Humans; Pharmacogenetics; Alleles; Computational Biology; High-Throughput Nucleotide Sequencing
PubMed: 38167765
DOI: 10.1093/jalm/jfad097 -
Nucleic Acids Research Nov 2023Small exons are pervasive in transcriptomes across organisms, and their quantification in RNA isoforms is crucial for understanding gene functions. Although long-read...
Small exons are pervasive in transcriptomes across organisms, and their quantification in RNA isoforms is crucial for understanding gene functions. Although long-read RNA-seq based on Oxford Nanopore Technologies (ONT) offers the advantage of covering transcripts in full length, its lower base accuracy poses challenges for identifying individual exons, particularly microexons (≤ 30 nucleotides). Here, we systematically assess small exons quantification in synthetic and human ONT RNA-seq datasets. We demonstrate that reads containing small exons are often not properly aligned, affecting the quantification of relevant transcripts. Thus, we develop a local-realignment method for misaligned exons (MisER), which remaps reads with misaligned exons to the transcript references. Using synthetic and simulated datasets, we demonstrate the high sensitivity and specificity of MisER for the quantification of transcripts containing small exons. Moreover, MisER enabled us to identify small exons with a higher percent spliced-in index (PSI) in neural, particularly neural-regulated microexons, when comparing 14 neural to 16 non-neural tissues in humans. Our work introduces an improved quantification method for long-read RNA-seq and especially facilitates studies using ONT long-reads to elucidate the regulation of genes involving small exons.
Topics: Humans; Exons; High-Throughput Nucleotide Sequencing; Protein Isoforms; RNA; RNA Isoforms; RNA-Seq; Sequence Analysis, RNA; Transcriptome
PubMed: 37843096
DOI: 10.1093/nar/gkad810 -
Nucleic Acids Research Jul 2023Ribosome profiling provides quantitative, comprehensive, and high-resolution snapshots of cellular translation by the high-throughput sequencing of short mRNA fragments...
Ribosome profiling provides quantitative, comprehensive, and high-resolution snapshots of cellular translation by the high-throughput sequencing of short mRNA fragments that are protected by ribosomes from nucleolytic digestion. While the overall principle is simple, the workflow of ribosome profiling experiments is complex and challenging, and typically requires large amounts of sample, limiting its broad applicability. Here, we present a new protocol for ultra-rapid ribosome profiling from low-input samples. It features a robust strategy for sequencing library preparation within one day that employs solid phase purification of reaction intermediates, allowing to reduce the input to as little as 0.1 pmol of ∼30 nt RNA fragments. Hence, it is particularly suited for the analyses of small samples or targeted ribosome profiling. Its high sensitivity and its ease of implementation will foster the generation of higher quality data from small samples, which opens new opportunities in applying ribosome profiling.
Topics: High-Throughput Nucleotide Sequencing; Protein Biosynthesis; Ribosome Profiling; Ribosomes; RNA, Messenger
PubMed: 37246712
DOI: 10.1093/nar/gkad459 -
Transplantation Oct 2023RNA-sequencing (RNA-seq) is a technique to determine the order of nucleotides in an RNA segment. Modern sequencing platforms simultaneously sequence millions of RNA...
RNA-sequencing (RNA-seq) is a technique to determine the order of nucleotides in an RNA segment. Modern sequencing platforms simultaneously sequence millions of RNA molecules. Advances in bioinformatics have allowed us to collect, store, analyze, and disseminate data from RNA-seq experiments and decipher biological insights from large sequencing datasets. Although bulk RNA-seq has significantly advanced our understanding of tissue-specific gene expression and regulation, recent advances in single-cell RNA-seq have allowed such information to be mapped to individual cells, thus remarkably enhancing our insight into discrete cellular functions within a biospecimen. These different RNA-seq experimental approaches require specialized computational tools. Herein, we will first review the RNA-seq experimental workflow, discuss the common terminologies used in RNA-seq, and suggest approaches for standardization across multiple studies. Next, we will provide an up-to-date appraisal of the applications of bulk RNA-seq and single-cell/nucleus RNA-seq in preclinical and clinical research on kidney transplantation, as well as typical bioinformatic workflows utilized in such analysis. Lastly, we will deliberate on the limitations of this technology in transplantation research and briefly summarize newer technologies that could be combined with RNA-seq to permit more powerful dissections of biological functions. Because each step in RNA-seq workflow has numerous variations and could potentially impact the results, as conscientious citizens of the research community, we must strive to continuously modernize our analytical pipelines and exhaustively report their technical details.
Topics: High-Throughput Nucleotide Sequencing; Sequence Analysis, RNA; Computational Biology; RNA; Reference Standards; Single-Cell Analysis
PubMed: 37026702
DOI: 10.1097/TP.0000000000004558 -
Scientific Reports Oct 2023Therapeutic antibody discovery often relies on in-vitro display methods to identify lead candidates. Assessing selected output diversity traditionally involves random...
Therapeutic antibody discovery often relies on in-vitro display methods to identify lead candidates. Assessing selected output diversity traditionally involves random colony picking and Sanger sequencing, which has limitations. Next-generation sequencing (NGS) offers a cost-effective solution with increased read depth, allowing a comprehensive understanding of diversity. Our study establishes NGS guidelines for antibody drug discovery, demonstrating its advantages in expanding the number of unique HCDR3 clusters, broadening the number of high affinity antibodies, expanding the total number of antibodies recognizing different epitopes, and improving lead prioritization. Surprisingly, our investigation into the correlation between NGS-derived frequencies of CDRs and affinity revealed a lack of association, although this limitation could be moderately mitigated by leveraging NGS clustering, enrichment and/or relative abundance across different regions to enhance lead prioritization. This study highlights NGS benefits, offering insights, recommendations, and the most effective approach to leverage NGS in therapeutic antibody discovery.
Topics: High-Throughput Nucleotide Sequencing; Antibodies; Epitopes
PubMed: 37884618
DOI: 10.1038/s41598-023-45538-w -
Nature Communications Oct 2023The short lengths of short-read sequencing reads challenge the analysis of paralogous genomic regions in exome and genome sequencing data. Most genetic variants within...
The short lengths of short-read sequencing reads challenge the analysis of paralogous genomic regions in exome and genome sequencing data. Most genetic variants within these homologous regions therefore remain unidentified in standard analyses. Here, we present a method (Chameleolyser) that accurately identifies single nucleotide variants and small insertions/deletions (SNVs/Indels), copy number variants and ectopic gene conversion events in duplicated genomic regions using whole-exome sequencing data. Application to a cohort of 41,755 exome samples yields 20,432 rare homozygous deletions and 2,529,791 rare SNVs/Indels, of which we show that 338,084 are due to gene conversion events. None of the SNVs/Indels are detectable using regular analysis techniques. Validation by high-fidelity long-read sequencing in 20 samples confirms >88% of called variants. Focusing on variation in known disease genes leads to a direct molecular diagnosis in 25 previously undiagnosed patients. Our method can readily be applied to existing exome data.
Topics: Humans; Exome; Polymorphism, Single Nucleotide; INDEL Mutation; DNA Copy Number Variations; Systems Analysis; High-Throughput Nucleotide Sequencing
PubMed: 37891200
DOI: 10.1038/s41467-023-42531-9 -
Journal of Visualized Experiments : JoVE Feb 2024AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification...
AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.
Topics: High-Throughput Nucleotide Sequencing; RNA; RNA, Transfer; Gene Library; DNA, Complementary; Sequence Analysis, RNA
PubMed: 38372376
DOI: 10.3791/66335 -
Methods in Molecular Biology (Clifton,... 2024The structure of RNA molecules is absolutely critical to their functions in a biological system. RNA structure is dynamic and changes in response to cellular needs....
The structure of RNA molecules is absolutely critical to their functions in a biological system. RNA structure is dynamic and changes in response to cellular needs. Within the last few decades, there has been an increased interest in studying the structure of RNA molecules and how they change to support the needs of the cell in different conditions. Selective 2'-hydroxyl acylation-based mutational profiling using high-throughput sequencing is a powerful method to predict the secondary structure of RNA molecules both in vivo and in immunopurified samples. Selective 2'-hydroxyl acylation-based mutational profiling using high-throughput sequencing works by adding bulky groups onto accessible "flexible" bases in an RNA molecule that are not involved in any base-pairing or RNA-protein interactions. When the RNA is reverse transcribed into cDNA, the bulky groups are incorporated as base mutations, which can be compared to an unmodified control to identify the locations of flexible bases. The comparison of sequence data between modified and unmodified samples allows the computer software program (developed to generate reactivity profiles) to generate RNA secondary structure models. These models can be compared in a variety of conditions to determine how specific stimuli influence RNA secondary structures.
Topics: RNA Folding; RNA; Mutation; High-Throughput Nucleotide Sequencing; Nucleic Acid Conformation; Software; Acylation
PubMed: 38907926
DOI: 10.1007/978-1-0716-3918-4_20 -
Annual Review of Biomedical Data Science Aug 2023The human microbiome is complex, variable from person to person, essential for health, and related to both the risk for disease and the efficacy of our treatments. There... (Review)
Review
The human microbiome is complex, variable from person to person, essential for health, and related to both the risk for disease and the efficacy of our treatments. There are robust techniques to describe microbiota with high-throughput sequencing, and there are hundreds of thousands of already-sequenced specimens in public archives. The promise remains to use the microbiome both as a prognostic factor and as a target for precision medicine. However, when used as an input in biomedical data science modeling, the microbiome presents unique challenges. Here, we review the most common techniques used to describe microbial communities, explore these unique challenges, and discuss the more successful approaches for biomedical data scientists seeking to use the microbiome as an input in their studies.
Topics: Humans; Microbiota; Precision Medicine; High-Throughput Nucleotide Sequencing
PubMed: 37159872
DOI: 10.1146/annurev-biodatasci-020722-043017 -
Cold Spring Harbor Protocols Oct 2023Transposon mutagenesis has been the method of choice for genetic screens and selections in bacteria by virtue of the transposon being linked to the disrupted gene,...
Transposon mutagenesis has been the method of choice for genetic screens and selections in bacteria by virtue of the transposon being linked to the disrupted gene, simplifying its identification. Transposon sequencing (Tn-seq) is a high-throughput version of transposon mutant screening, in which massively parallel sequencing is used to simultaneously follow the fitness of all mutants in a complex library. In a single experiment, one can use Tn-seq to interrogate the contribution of all genes of a bacterium to fitness under a condition of interest. Here, we introduce a method to construct a saturating transposon insertion library in Gram-negative bacteria, to capture the transposon junctions , and to identify essential genes and conditional genes using massively parallel sequencing. The accompanying protocol was developed as part of Cold Spring Harbor's Advanced Bacterial Genetics course.
Topics: Mutagenesis, Insertional; DNA Transposable Elements; High-Throughput Nucleotide Sequencing; Gene Library
PubMed: 36931734
DOI: 10.1101/pdb.top107867