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September 22, 2019

Metataxonomic and metagenomic approaches vs. culture-based techniques for clinical pathology.

Diagnoses that are both timely and accurate are critically important for patients with life-threatening or drug resistant infections. Technological improvements in High-Throughput Sequencing (HTS) have led to its use in pathogen detection and its application in clinical diagnoses of infectious diseases. The present study compares two HTS methods, 16S rRNA marker gene sequencing (metataxonomics) and whole metagenomic shotgun sequencing (metagenomics), in their respective abilities to match the same diagnosis as traditional culture methods (culture inference) for patients with ventilator associated pneumonia (VAP). The metagenomic analysis was able to produce the same diagnosis as culture methods at the species-level for five of the six samples, while the metataxonomic analysis was only able to produce results with the same species-level identification as culture for two of the six samples. These results indicate that metagenomic analyses have the accuracy needed for a clinical diagnostic tool, but full integration in diagnostic protocols is contingent on technological improvements to decrease turnaround time and lower costs.

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September 22, 2019

Next-generation approaches to advancing eco-immunogenomic research in critically endangered primates.

High-throughput sequencing platforms are generating massive amounts of genomic data from nonmodel species, and these data sets are valuable resources that can be mined to advance a number of research areas. An example is the growing amount of transcriptome data that allow for examination of gene expression in nonmodel species. Here, we show how publicly available transcriptome data from nonmodel primates can be used to design novel research focused on immunogenomics. We mined transcriptome data from the world’s most endangered group of primates, the lemurs of Madagascar, for sequences corresponding to immunoglobulins. Our results confirmed homology between strepsirrhine and haplorrhine primate immunoglobulins and allowed for high-throughput sequencing of expressed antibodies (Ig-seq) in Coquerel’s sifaka (Propithecus coquereli). Using both Pacific Biosciences RS and Ion Torrent PGM sequencing, we performed Ig-seq on two individuals of Coquerel’s sifaka. We generated over 150 000 sequences of expressed antibodies, allowing for molecular characterization of the antigen-binding region. Our analyses suggest that similar VDJ expression patterns exist across all primates, with sequences closely related to the human VH 3 immunoglobulin family being heavily represented in sifaka antibodies. Moreover, the antigen-binding region of sifaka antibodies exhibited similar amino acid variation with respect to haplorrhine primates. Our study represents the first attempt to characterize sequence diversity of the expressed antibody repertoire in a species of lemur. We anticipate that methods similar to ours will provide the framework for investigating the adaptive immune response in wild populations of other nonmodel organisms and can be used to advance the burgeoning field of eco-immunology. © 2014 John Wiley & Sons Ltd.

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September 22, 2019

Revealing missing human protein isoforms based on Ab initio prediction, RNA-seq and proteomics.

Biological and biomedical research relies on comprehensive understanding of protein-coding transcripts. However, the total number of human proteins is still unknown due to the prevalence of alternative splicing. In this paper, we detected 31,566 novel transcripts with coding potential by filtering our ab initio predictions with 50 RNA-seq datasets from diverse tissues/cell lines. PCR followed by MiSeq sequencing showed that at least 84.1% of these predicted novel splice sites could be validated. In contrast to known transcripts, the expression of these novel transcripts were highly tissue-specific. Based on these novel transcripts, at least 36 novel proteins were detected from shotgun proteomics data of 41 breast samples. We also showed L1 retrotransposons have a more significant impact on the origin of new transcripts/genes than previously thought. Furthermore, we found that alternative splicing is extraordinarily widespread for genes involved in specific biological functions like protein binding, nucleoside binding, neuron projection, membrane organization and cell adhesion. In the end, the total number of human transcripts with protein-coding potential was estimated to be at least 204,950.

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September 22, 2019

Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development.

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September 22, 2019

G&T-seq: parallel sequencing of single-cell genomes and transcriptomes.

The simultaneous sequencing of a single cell’s genome and transcriptome offers a powerful means to dissect genetic variation and its effect on gene expression. Here we describe G&T-seq, a method for separating and sequencing genomic DNA and full-length mRNA from single cells. By applying G&T-seq to over 220 single cells from mice and humans, we discovered cellular properties that could not be inferred from DNA or RNA sequencing alone.

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September 22, 2019

Shorter unreported sequences in a RACE-Seq study involving seven tissues confirms ~150 novel transcripts identified in MCF-7 cell line PacBio transcriptome, leaving ~100 non-redundant transcripts exclusive to the cancer cell line.

PacBio sequencing generates much longer reads compared to second-generation sequencing technologies, with a trade-off of lower throughput, higher error rate and more cost per base. The PacBio transcriptome of the breast cancer cell line MCF-7 was found to have ~300 transcripts un-annotated in the current GENCODE (v25) or RefSeq, and missing in the liver, heart and brain PacBio transcriptomes [1]. RACE-sequencing (RACE-seq [2]) extends a well-established method of characterizing cDNA molecules generated by rapid amplification of cDNA ends (RACE [3]) using high-throughput sequencing technologies, reducing costs compared to PacBio. Here, shorter fragments of ~150 transcripts were found to be present in seven tissues analyzed in a recent RACE-seq study (Accid:ERP012249) [4]. These transcripts were not among the ~2500 novel transcripts reported in that study, tested separately here using the genomic coordinates provided, although “all curated novel isoforms were incorporated into the human GENCODE set (v22)” in that study. Non-redundancy analysis of the exclusive transcripts identified one transcript mapping to Chr1 with seven different splice variants, and erroneously mapped to Chr15 (PAC clone 15q11-q13) from the Prader-Willi/Angelman Syndrome region (Accid:AC004137.1). Finally, there are ~100 non-redundant transcripts missing in the seven tissues, in addition to other three tissues analyzed previously. Their absence in GENCODE and RefSeq databases rule them out as commonly transcribed regions, further increasing their likelihood as biomarkers.

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September 22, 2019

Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing.

The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing.In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells.Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

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September 22, 2019

Elevated expression of a minor isoform of ANK3 is a risk factor for bipolar disorder.

Ankyrin-3 (ANK3) is one of the few genes that have been consistently identified as associated with bipolar disorder by multiple genome-wide association studies. However, the exact molecular basis of the association remains unknown. A rare loss-of-function splice-site SNP (rs41283526*G) in a minor isoform of ANK3 (incorporating exon ENSE00001786716) was recently identified as protective of bipolar disorder and schizophrenia. This suggests that an elevated expression of this isoform may be involved in the etiology of the disorders. In this study, we used novel approaches and data sets to test this hypothesis. First, we strengthen the statistical evidence supporting the allelic association by replicating the protective effect of the minor allele of rs41283526 in three additional large independent samples (meta-analysis p-values: 6.8E-05 for bipolar disorder and 8.2E-04 for schizophrenia). Second, we confirm the hypothesis that both bipolar and schizophrenia patients have a significantly higher expression of this isoform than controls (p-values: 3.3E-05 for schizophrenia and 9.8E-04 for bipolar type I). Third, we determine the transcription start site for this minor isoform by Pacific Biosciences sequencing of full-length cDNA and show that it is primarily expressed in the corpus callosum. Finally, we combine genotype and expression data from a large Norwegian sample of psychiatric patients and controls, and show that the risk alleles in ANK3 identified by bipolar disorder GWAS are located near the transcription start site of this isoform and are significantly associated with its elevated expression. Together, these results point to the likely molecular mechanism underlying ANK3´s association with bipolar disorder.

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September 22, 2019

Single-molecule DNA sequencing of acute myeloid leukemia and myelodysplastic syndromes with multiple TP53 alterations.

Although the frequency of TP53 mutations in hemato- logic malignancies is low, these mutations have a high clinical relevance and are usually associated with poor prognosis. Somatic TP53 mutations have been detected in up to 73.3% of cases of acute myeloid leukemia (AML) with complex karyotype and 18.9% of AML with other unfavorable cytogenetic risk factors. AML with TP53 mutations, and/or chromosomal aneuploidy, has been defined as a distinct AML subtype. In low-risk myelodysplastic syndromes (MDS), TP53 mutations occur at an early disease stage and predict disease progression. TP53 mutation diagnosis is now part of the revised European LeukemiaNet (ELN) guidelines.

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September 22, 2019

Next generation multilocus sequence typing (NGMLST) and the analytical software program MLSTEZ enable efficient, cost-effective, high-throughput, multilocus sequencing typing.

Multilocus sequence typing (MLST) has become the preferred method for genotyping many biological species, and it is especially useful for analyzing haploid eukaryotes. MLST is rigorous, reproducible, and informative, and MLST genotyping has been shown to identify major phylogenetic clades, molecular groups, or subpopulations of a species, as well as individual strains or clones. MLST molecular types often correlate with important phenotypes. Conventional MLST involves the extraction of genomic DNA and the amplification by PCR of several conserved, unlinked gene sequences from a sample of isolates of the taxon under investigation. In some cases, as few as three loci are sufficient to yield definitive results. The amplicons are sequenced, aligned, and compared by phylogenetic methods to distinguish statistically significant differences among individuals and clades. Although MLST is simpler, faster, and less expensive than whole genome sequencing, it is more costly and time-consuming than less reliable genotyping methods (e.g. amplified fragment length polymorphisms). Here, we describe a new MLST method that uses next-generation sequencing, a multiplexing protocol, and appropriate analytical software to provide accurate, rapid, and economical MLST genotyping of 96 or more isolates in single assay. We demonstrate this methodology by genotyping isolates of the well-characterized, human pathogenic yeast Cryptococcus neoformans. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

High-resolution comparative analysis of great ape genomes.

Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single- to mega-base pair-sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these variants are enriched near genes that are down-regulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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September 22, 2019

Extensive allele-specific translational regulation in hybrid mice.

Translational regulation is mediated through the interaction between diffusible trans-factors and cis-elements residing within mRNA transcripts. In contrast to extensively studied transcriptional regulation, cis-regulation on translation remains underexplored. Using deep sequencing-based transcriptome and polysome profiling, we globally profiled allele-specific translational efficiency for the first time in an F1 hybrid mouse. Out of 7,156 genes with reliable quantification of both alleles, we found 1,008 (14.1%) exhibiting significant allelic divergence in translational efficiency. Systematic analysis of sequence features of the genes with biased allelic translation revealed that local RNA secondary structure surrounding the start codon and proximal out-of-frame upstream AUGs could affect translational efficiency. Finally, we observed that the cis-effect was quantitatively comparable between transcriptional and translational regulation. Such effects in the two regulatory processes were more frequently compensatory, suggesting that the regulation at the two levels could be coordinated in maintaining robustness of protein expression. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

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September 22, 2019

Genome-wide characterization of human L1 antisense promoter-driven transcripts.

Long INterspersed Element-1 (LINE-1 or L1) is the only autonomously active, transposable element in the human genome. L1 sequences comprise approximately 17 % of the human genome, but only the evolutionarily recent, human-specific subfamily is retrotransposition competent. The L1 promoter has a bidirectional orientation containing a sense promoter that drives the transcription of two proteins required for retrotransposition and an antisense promoter. The L1 antisense promoter can drive transcription of chimeric transcripts: 5′ L1 antisense sequences spliced to the exons of neighboring genes.The impact of L1 antisense promoter activity on cellular transcriptomes is poorly understood. To investigate this, we analyzed GenBank ESTs for messenger RNAs that initiate in the L1 antisense promoter. We identified 988 putative L1 antisense chimeric transcripts, 911 of which have not been previously reported. These appear to be alternative genic transcripts, sense-oriented with respect to gene and initiating near, but typically downstream of, the gene transcriptional start site. In multiple cell lines, L1 antisense promoters display enrichment for YY1 transcription factor and histone modifications associated with active promoters. Global run-on sequencing data support the activity of the L1 antisense promoter. We independently detected 124 L1 antisense chimeric transcripts using long read Pacific Biosciences RNA-seq data. Furthermore, we validated four chimeric transcripts by quantitative RT-PCR and Sanger sequencing and demonstrated that they are readily detectable in many normal human tissues.We present a comprehensive characterization of human L1 antisense promoter-driven transcripts and provide substantial evidence that they are transcribed in a variety of human cell-types. Our findings reveal a new wide-reaching aspect of L1 biology by identifying antisense transcripts affecting as many as 4 % of all human genes.

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September 22, 2019

A single-molecule long-read survey of the human transcriptome.

Global RNA studies have become central to understanding biological processes, but methods such as microarrays and short-read sequencing are unable to describe an entire RNA molecule from 5′ to 3′ end. Here we use single-molecule long-read sequencing technology from Pacific Biosciences to sequence the polyadenylated RNA complement of a pooled set of 20 human organs and tissues without the need for fragmentation or amplification. We show that full-length RNA molecules of up to 1.5 kb can readily be monitored with little sequence loss at the 5′ ends. For longer RNA molecules more 5′ nucleotides are missing, but complete intron structures are often preserved. In total, we identify ~14,000 spliced GENCODE genes. High-confidence mappings are consistent with GENCODE annotations, but >10% of the alignments represent intron structures that were not previously annotated. As a group, transcripts mapping to unannotated regions have features of long, noncoding RNAs. Our results show the feasibility of deep sequencing full-length RNA from complex eukaryotic transcriptomes on a single-molecule level.

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September 22, 2019

Evaluation of tools for long read RNA-seq splice-aware alignment.

High-throughput sequencing has transformed the study of gene expression levels through RNA-seq, a technique that is now routinely used by various fields, such as genetic research or diagnostics. The advent of third generation sequencing technologies providing significantly longer reads opens up new possibilities. However, the high error rates common to these technologies set new bioinformatics challenges for the gapped alignment of reads to their genomic origin. In this study, we have explored how currently available RNA-seq splice-aware alignment tools cope with increased read lengths and error rates. All tested tools were initially developed for short NGS reads, but some have claimed support for long Pacific Biosciences (PacBio) or even Oxford Nanopore Technologies (ONT) MinION reads.The tools were tested on synthetic and real datasets from two technologies (PacBio and ONT MinION). Alignment quality and resource usage were compared across different aligners. The effect of error correction of long reads was explored, both using self-correction and correction with an external short reads dataset. A tool was developed for evaluating RNA-seq alignment results. This tool can be used to compare the alignment of simulated reads to their genomic origin, or to compare the alignment of real reads to a set of annotated transcripts. Our tests show that while some RNA-seq aligners were unable to cope with long error-prone reads, others produced overall good results. We further show that alignment accuracy can be improved using error-corrected reads.https://github.com/kkrizanovic/RNAseqEval, https://figshare.com/projects/RNAseq_benchmark/24391.mile.sikic@fer.hr.Supplementary data are available at Bioinformatics online.© The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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