De Novo Assembly And Analysis Of Rna Seq Data PdfBy Theresa O. In and pdf 21.05.2021 at 04:58 9 min read
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- Tutorial 5: RNA-Seq de novo transcriptome workflow
- De Novo Sequencing
- Evaluation of de novo transcriptome assemblies from RNA-Seq data
Available as a PDF tutorial. This tutorial will show you how to link variants to positions on a 3D protein structure, and how to interpret the resulting interactive 3D model. The focus will be on identifying variants associated with drug resistance to chronic myeloid leukemia treatment. Use the tools and functionalities of the workbench to simplify your cloning strategy and visualize every steps of the process: Look for restriction enzymes, design primers, and simulate your cloning strategy and results. A guide to the most fundamental functionalities of your workbench: Learn how to import data in the workbench, how to run a tool and use the toolbar and side panels settings to visualize your results in different ways.
Tutorial 5: RNA-Seq de novo transcriptome workflow
For the organisms without the reference transcriptome, de novo transcriptome assembly must be carried out prior to quantification. However, a large number of erroneous contigs produced by the assemblers might result in unreliable estimation.
In this regard, this study investigates how assembly quality affects the performance of quantification based on de novo transcriptome assembly. We examined the over-extended and incomplete contigs, and demonstrated that assembly completeness has a strong impact on the estimation of contig abundance. Then we investigated the behavior of the quantifiers with respect to sequence ambiguity which might be originally presented in the transcriptome or accidentally produced by assemblers.
The results suggested that the quantifiers often over-estimate the expression of family-collapse contigs and under-estimate the expression of duplicated contigs. For organisms without reference transcriptome, it remains challenging to detect the inaccurate estimation on family-collapse contigs.
On the contrary, we observed that the situation of under-estimation on duplicated contigs can be warned through analyzing the read proportion of estimated abundance RPEA of contigs in the connected component inferenced by the quantifiers.
In addition, we suggest that the estimated quantification results on the connected component level have better accuracy over sequence level quantification.
The analytic results conducted in this study provides valuable insights for future development of transcriptome assembly and quantification. Quantification and comparison of transcript expression are essential to understanding the role of RNA in different physiological conditions or developmental stages. Such experiments and analyses are widely used in the studies of molecular biology. Over the past decades, several biological technologies have been developed to quantify the abundance of transcripts, such as expression microarray 1 and high-throughput RNA sequencing RNA-Seq 2.
For organisms with sufficient genomic information, the design of microarray provides a high throughput and cost-effective solution to examine transcript expression. On the other hand, RNA-Seq is superior in delivering lower background signals and larger dynamic ranges 3.
Despite the fact that many genome sequencing projects have been carried out, such as Genome10K 4 , arthropod genomes initiative i5K 5 and Bird10K 6 , whole genome studies are still demanding efforts for many research groups. For non-model organisms, the expression microarray needs to rely on cross-species hybridization 7. On the contrary, RNA-Seq is more suitable owing to its capability of detecting novel transcripts without additional genomic information 3.
When the reference genome and transcriptome are not available, RNA-Seq reads are first used to reconstruct the transcriptome 8 , 9. These methods are able to inference the abundance of expression without the need of genomic sequences, using the number of RNA-Seq reads that overlap with the assembled contigs 9.
Nevertheless, quantification is much more challenging without reliable reference sequences because of the erroneous contigs produced by the assemblers, which often result from sequencing errors, insufficient sequencing depth and biological variability To address these problems, a great number of comparative studies have been published recently.
While many studies evaluated transcriptome assembly 19 , 20 , 21 or quantification 22 , 23 programs independently, few have discussed how transcriptome assembly influences the downstream quantification analysis. In , Vijay, N. That study examined the impact of various aspects of sequencing reads on transcriptome assembly and differentially expressed genes DEG analysis 7 , but the effect of redundant contigs and multiple-mapping reads on quantification was not well discussed.
Another study conducted by Wang, S. Gribskov evaluated the quality of assembled contigs and their effects on DEG analysis However, their study mainly focused on the evaluation of entire workflow from assembly, quantification, to DEG analysis, which makes it obscure to unravel how the erroneous contigs affect the authenticity of downstream analysis. In addition, some studies investigated the reliability of quantification algorithms by utilizing the information regarding splicing junctions.
For example, in , Soneson, C. Afterwards, Cong Ma, et al. It should be noticed that the implementation of these ideas requires proper annotation of the genome, such as the splicing junctions and coordinates of untranslated regions UTR for the transcripts. In this regard, it remains challenging to analyze the quantification reliability of the assembled contigs generated from de novo assembly without proper annotation of the genome.
In this study, we used both in silico simulated and experimental RNA-Seq data from three species yeast, dog, and mouse. After that, the assembled contigs were evaluated based on TransRate 19 scores, which were previously proposed to assess the quality of de novo transcriptome assemblies using the alignments of sequencing reads to the assembled sequences.
After de novo assembly, the reference transcripts were assigned to assembled contigs according to the BLASTn 27 alignments. Each transcript-contig alignment was then categorized based on accuracy, recovery and sequence ambiguity. By exploring the interplay between each stage in RNA-Seq analysis workflow, this study provides valuable insights into conducting RNA-Seq analysis and we anticipate these discoveries would be useful in the future development of assembly or quantification algorithms.
Three experimental and three simulated RNA-Seq datasets were used in this study. Both experimental and simulated data included three species: yeast Saccharomyces cerevisiae , dog Canis lupus familiaris and mouse Mus musculus. The yeast dataset SRR was from the study of Nookaew et al. The dog dataset SRR was produced by Liu, et al. Finally, the mouse dataset SRR was collected from the study of Grabherr, et al.
For the simulated datasets, Flux Simulator ver. To facilitate the analysis, only the transcripts annotated as messenger RNA mRNA and with over nucleotides in length were extracted. In total, The quality of both experimental and simulated datasets was examined using FastQC ver. The resultant RNA reads that were unable to maintain the paired relation were discarded. To ensure the conclusions drawn in this study are consistent across different sequencing depths, we created additional datasets with a higher sequencing depth.
For yeast, we adopted two additional datasets SRR and SRR, which are the biological replicates of the yeast data we used SRR , to create a new dataset with a high-sequencing depth, denoted as the experimental H yeast dataset. As for the experimental H dog dataset, we adopted another dataset SRR, which has a higher sequencing depth in the same research of the experimental dog dataset SRR In order to evaluate the performance of transcript quantification, the ground truth of expression abundance for each transcript must be first determined.
For simulated datasets, the number of the generated RNA reads for each transcript was recorded during the simulation process. Since transcriptome assemblers sometimes generate duplicated, incomplete or over-extended contigs, the metrics we use for quantifying expression must consider the normalization with respect to both sequence length and the number of total nucleotides.
In contrast, because the ground truth abundance for each RNA molecule is unknown for experimental datasets, we calculated the average TPM inferred by Kallisto ver. Although the estimated expression might not perfectly reflect the real number of RNA molecules in a biological sample, it still provides valuable information when comparing the performance of quantification before and after de novo transcriptome assembly.
To minimize the effect of fragmented contigs, only the contigs with over nucleotides in length were kept for the quantification analysis. The assemblies were evaluated based on the length of contigs, the number of recovered transcripts, the number of erroneous contigs and the evaluation scores provided by TransRate. The TransRate scores that we used in this study are the score of bases covered , score of good mapping , score of not segmented and overall score. The score of bases covered represents the proportion of nucleotide bases in a contig that are covered by reads.
The score of good mapping represents the proportion of read pairs of which both reads are aligned in the correct orientation on a single contig. The score of not segmented represents the proportion of contigs that might be a chimera of multiple transcripts.
Subsequently, the expression abundance for each contig was estimated using one alignment-based and two alignment-free quantifiers, namely 1 Bowtie2 ver. For the purpose of comparing the estimated abundance of contigs with the ground truth expression from the corresponding transcripts, we assigned the reference transcripts cDNA to assembled contigs based on BLASTn 2.
We integrated the remained HSPs onto the coordinates of both transcript and contig to obtain the global alignment. Similar to a previous study 7 , we calculated the recovery and accuracy for each global alignment, which refer to the proportion of matched nucleotides on the transcript and the proportion of correctly matched nucleotides on the contig respectively Supplementary File 2: Fig.
In this manner, we were able to identify all the corresponding transcripts for each contig. Note that it is possible that a contig can be associated with multiple transcripts, and a transcript can assign to multiple contigs as well. We considered multiple assignments here in order to understand the impact of redundant sequences on the quantification. Once the transcripts have been assigned to the contigs, we used Eq. To determine the origin of the RNA-Seq reads that can be mapped to multiple transcripts is an important issue for the development of quantification algorithms.
In this regard, it is of interest to understand the impact of sequence ambiguity on transcript quantification. The size of a connected component is defined as the number of sequence members inside. Furthermore, we used the read proportion of estimated abundance RPEA of a contig in a connected component to investigate the behavior of quantifier while ambiguous sequences are presented. Construction of Ambiguity Network.
The diagram illustrates how pairwise alignments in a contig set are employed to construct ambiguity networks. The ambiguity network is first initialized by given the contig sequences, creating a single cluster for each sequence. In this study, the ambiguity network can be constructed for both contig and transcript sets. For the purpose of simplicity, we only illustrated the scenario for contigs in this figure.
If the highest RPEA in the connected component is close to 1, it suggests that the quantifier allocates all the reads in the connected component to one specific contig. The assembled contigs are categorized into five particular categories in this study: 1 full-length , 2 incompleteness , 3 over-extension , 4 family-collapse and 5 duplication Fig. The contigs identified as incompleteness , over-extension , family-collapse and duplication are called erroneous contigs throughout this study.
The analysis of the first three categories were not affected by the factor of sequence ambiguity, allowing us to investigate the impact of assembly completeness on quantification independently.
Given the length of contig l c and the length of the corresponding transcript l t , the assembly completeness of a contig was examined through the difference in length:. Examples of Contig Categories. The diagram gives examples for each contig category we analyzed throughout this study. The middle column shows an example for each category, while the right column portrays the relation of contigs and transcripts in network representation.
The sequence nodes are connected together in solid line if they are in the same ambiguity cluster. On the other hand, a blue dot arrow represents the transcript assignment for the contigs.
The analysis of contigs labeled as full-length, incompleteness and over-extended exclude the factor of sequence ambiguity. In contrast, family-collapse and duplication remove the potential effect of assembly completeness, focusing only on the impact of redundant or duplicated sequences. To be more specific, family-collapse represents contigs which are assigned with multiple transcripts and duplication stands for the multiple contigs assigned by a single transcript.
By examining these contigs, the problems caused by the assemblers that fail to distinguish similar transcripts from each other or generate a large number of redundant contigs were investigated.
De Novo Sequencing
Skip to search form Skip to main content You are currently offline. Some features of the site may not work correctly. DOI: Collins and J. Thomson and R. Stewart and Colin N.
De novo transcriptome assembly is the de novo sequence assembly method of creating a transcriptome without the aid of a reference genome. As a result of the development of novel sequencing technologies, the years between and saw a large drop in the cost of sequencing. Examining non-model organisms can provide novel insights into the mechanisms underlying the "diversity of fascinating morphological innovations" that have enabled the abundance of life on planet Earth. De novo transcriptome assembly is often the preferred method to studying non-model organisms, since it is cheaper and easier than building a genome, and reference-based methods are not possible without an existing genome. The transcriptomes of these organisms can thus reveal novel proteins and their isoforms that are implicated in such unique biological phenomena. A set of assembled transcripts allows for initial gene expression studies.
Some genome assemblers have already succeeded in as- sembling transcriptome from RNA-seq data [6,7]. However, different from genome assembly, there are.
Evaluation of de novo transcriptome assemblies from RNA-Seq data
Since no reference genome exists, we utilized Trinity, an RNA-seq de novo transcriptome assembler, in order to reconstruct full-length transcripts and alternatively spliced isoforms from our RNA-seq data. A total of , transcripts were assembled with a N50 contig length of 1, We then aligned our RNA-seq data to the assembled transcriptome using Bowtie to assess the quality of the assembly. The expression estimates generated by RSEM from both our assembled transcriptome and a reference transcriptome were used with two differential expression analysis tools, edgeR and DESeq, in order to determine which genes are being differentially expressed in PPO-silenced plants as compared to wild-type. Using DESeq, we discovered 69 genes from our assembled transcriptome and 46 genes from the reference transcriptome to be differentially expressed based on an adjusted p-value of 0.
Protocol DOI: De novo assembly of RNA-seq data enables researchers to study transcriptomes without the need for a genome sequence; this approach can be usefully applied, for instance, in research on 'non-model organisms' of ecological and evolutionary importance,. De novo assembly of RNA-seq data enables researchers to study transcriptomes without the need for a genome sequence; this approach can be usefully applied, for instance, in research on 'non-model organisms' of ecological and evolutionary importance, cancer samples or the microbiome. In this protocol we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-seq data in non-model organisms.
The nematode Ascaridia galli order Ascaridida is an economically important intestinal parasite responsible for increased food consumption, reduced performance and elevated mortality in commercial poultry production. This roundworm is an emerging problem in several European countries on farms with laying hens, as a consequence of the recent European Union EU ban on conventional battery cages. As infection is associated with slow development of low levels of acquired protective immunity, parasite control relies on repeated use of dewormers anthelmintics. Thus we developed a reference transcriptome of A. Transcriptional variations between treated and untreated A.
De novo sequencing refers to sequencing a novel genome where there is no reference sequence available for alignment. Sequence reads are assembled as contigs, and the coverage quality of de novo sequence data depends on the size and continuity of the contigs ie, the number of gaps in the data. Next-generation sequencing NGS allows faster, more accurate characterization of any species compared to traditional methods, such as Sanger sequencing. Illumina NGS technology offers rapid, comprehensive, accurate characterization of any species. View Recommended Workflow. When sequencing a genome for the first time, a combined approach can yield higher-quality assemblies.
Metrics details. De novo assembly of RNA-seq data allows the study of transcriptome in absence of a reference genome either if data is obtained from a single organism or from a mixed sample as in metatranscriptomics studies. Given the high number of sequences obtained from NGS approaches, a critical step in any analysis workflow is the assembly of reads to reconstruct transcripts thus reducing the complexity of the analysis. Despite many available tools show a good sensitivity, there is a high percentage of false positives due to the high number of assemblies considered and it is likely that the high frequency of false positive is underestimated by currently used benchmarks. Moreover, benchmarks performed are usually based on RNA-seq data from annotated genomes and assembled transcripts are compared to annotations and genomes to identify putative good and wrong reconstructions, but these tests alone may lead to accept a particular type of false positive as true, as better described below. Here we present a novel methodology of de novo assembly, implemented in a software named STAble Short-reads Transcriptome Assembler. The novel concept of this assembler is that the whole reads are used to determine possible alignments instead of using smaller k-mers, with the aim of reducing the number of chimeras produced.
In this tutorial, you will de novo assemble an abbreviated set of paired end RNA -Seq sequences from Saccharomyces cerevisiae yeast from Nookaew I et al. This workflow uses an abbreviated yeast data set with about 1 million reads per file. With other applications, de novo assembly of RNA -Seq data can potentially result in thousands of unlabeled contigs representing the expressed transcripts. Results from this workflow are non-quantitative. The Identified Transcripts tab is active, by default. You should see over Total Identified Transcripts. Need more help with this?
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