Rainbowfish: A Succinct Colored de Bruijn Graph Representation

AbstractThe colored de Bruijn graph— a variant of the de Bruijn graph which associates each edge (i.e., k-mer) with some set of colors — is an increasingly important combinatorial structure in computational biology. Iqbal et al. demonstrated the utility of this structure for representing and assembling a collection (pop-ulation) of genomes, and showed how it can be used to accurately detect genetic variants. Muggli et al. introduced VARI, a representation of the colored de Bruijn graph that adopts the BOSS representation for the de Bruijn graph topology and achieves considerable savings in space over Cortex, albeit with some sacrifice in speed. The memory-efficient representation of VARI allows the colored de Bruijn graph to be constructed and analyzed for large datasets, beyond what is possible with Cortex.In this paper, we introduce Rainbowfish, a succinct representation of the color information of the colored de Bruijn graph that reduces the space usage even further. Our representation also uses BOSS to represent the de Bruijn graph, but decomposes the color sets based on an equivalence relation and exploits the inherent skewness in the distribution of these color sets. The Rainbowfish representation is compressed based on the 0th-order entropy of the color sets, which can lead to a significant reduction in the space required to store the relevant information for each edge. In practice, Rainbowfish achieves up to a 20 × improvement in space over VARI. Rainbowfish is written in C++11 and is available at https://github.com/COMBINE-lab/rainbowfish.</jats:p

Perplexity: Evaluating Transcript Abundance Estimation in the Absence of Ground Truth

There has been rapid development of probabilistic models and inference methods for transcript abundance estimation from RNA-seq data. These models aim to accurately estimate transcript-level abundances, to account for different biases in the measurement process, and even to assess uncertainty in resulting estimates that can be propagated to subsequent analyses. The assumed accuracy of the estimates inferred by such methods underpin gene expression based analysis routinely carried out in the lab. Although hyperparameter selection is known to affect the distributions of inferred abundances (e.g. producing smooth versus sparse estimates), strategies for performing model selection in experimental data have been addressed informally at best. Thus, we derive perplexity for evaluating abundance estimates on fragment sets directly. We adapt perplexity from the analogous metric used to evaluate language and topic models and extend the metric to carefully account for corner cases unique to RNA-seq. In experimental data, estimates with the best perplexity also best correlate with qPCR measurements. In simulated data, perplexity is well behaved and concordant with genome-wide measurements against ground truth and differential expression analysis. To our knowledge, our study is the first to make possible model selection for transcript abundance estimation on experimental data in the absence of ground truth

Swimming downstream: statistical analysis of differential transcript usage following Salmon quantification

Detection of differential transcript usage (DTU) from RNA-seq data is an important bioinformatic analysis that complements differential gene expression analysis. Here we present a simple workflow using a set of existing R/Bioconductor packages for analysis of DTU. We show how these packages can be used downstream of RNA-seq quantification using the Salmon software package. The entire pipeline is fast, benefiting from inference steps by Salmon to quantify expression at the transcript level. The workflow includes live, runnable code chunks for analysis using DRIMSeq and DEXSeq, as well as for performing two-stage testing of DTU using the stageR package, a statistical framework to screen at the gene level and then confirm which transcripts within the significant genes show evidence of DTU. We evaluate these packages and other related packages on a simulated dataset with parameters estimated from real data

Where the patterns are: repetition-aware compression for colored de Bruijn graphs

We describe lossless compressed data structures for the colored de Bruijn graph (or c-dBG). Given a collection of reference sequences, a c-dBG can be essentially regarded as a map from k-mers to their color sets. The color set of a k-mer is the set of all identifiers, or colors, of the references that contain the k-mer. While these maps find countless applications in computational biology (e.g., basic query, reading mapping, abundance estimation, etc.), their memory usage represents a serious challenge for large-scale sequence indexing. Our solutions leverage on the intrinsic repetitiveness of the color sets when indexing large collections of related genomes. Hence, the described algorithms factorize the color sets into patterns that repeat across the entire collection and represent these patterns once instead of redundantly replicating their representation as would happen if the sets were encoded as atomic lists of integers. Experimental results across a range of datasets and query workloads show that these representations substantially improve over the space effectiveness of the best previous solutions (sometimes, even dramatically, yielding indexes that are smaller by an order of magnitude). Despite the space reduction, these indexes only moderately impact the efficiency of the queries compared to the fastest indexes

Fast, Parallel, and Cache-Friendly Suffix Array Construction

String indexes such as the suffix array (SA) and the closely related longest common prefix (LCP) array are fundamental objects in bioinformatics and have a wide variety of applications. Despite their importance in practice, few scalable parallel algorithms for constructing these are known, and the existing algorithms can be highly non-trivial to implement and parallelize. In this paper we present CaPS-SA, a simple and scalable parallel algorithm for constructing these string indexes inspired by samplesort. Due to its design, CaPS-SA has excellent memory-locality and thus incurs fewer cache misses and achieves strong performance on modern multicore systems with deep cache hierarchies. We show that despite its simple design, CaPS-SA outperforms existing state-of-the-art parallel SA and LCP-array construction algorithms on modern hardware. Finally, motivated by applications in modern aligners where the query strings have bounded lengths, we introduce the notion of a bounded-context SA and show that CaPS-SA can easily be extended to exploit this structure to obtain further speedups

TransRate: reference-free quality assessment of de novo transcriptome assemblies.

TransRate is a tool for reference-free quality assessment of de novo transcriptome assemblies. Using only the sequenced reads and the assembly as input, we show that multiple common artifacts of de novo transcriptome assembly can be readily detected. These include chimeras, structural errors, incomplete assembly, and base errors. TransRate evaluates these errors to produce a diagnostic quality score for each contig, and these contig scores are integrated to evaluate whole assemblies. Thus, TransRate can be used for de novo assembly filtering and optimization as well as comparison of assemblies generated using different methods from the same input reads. Applying the method to a data set of 155 published de novo transcriptome assemblies, we deconstruct the contribution that assembly method, read length, read quantity, and read quality make to the accuracy of de novo transcriptome assemblies and reveal that variance in the quality of the input data explains 43% of the variance in the quality of published de novo transcriptome assemblies. Because TransRate is reference-free, it is suitable for assessment of assemblies of all types of RNA, including assemblies of long noncoding RNA, rRNA, mRNA, and mixed RNA samples

Fulgor: a fast and compact k-mer index for large-scale matching and color queries

The problem of sequence identification or matching - determining the subset of reference sequences from a given collection that are likely to contain a short, queried nucleotide sequence - is relevant for many important tasks in Computational Biology, such as metagenomics and pangenome analysis. Due to the complex nature of such analyses and the large scale of the reference collections a resource-efficient solution to this problem is of utmost importance. This poses the threefold challenge of representing the reference collection with a data structure that is efficient to query, has light memory usage, and scales well to large collections. To solve this problem, we describe an efficient colored de Bruijn graph index, arising as the combination of a k-mer dictionary with a compressed inverted index. The proposed index takes full advantage of the fact that unitigs in the colored compacted de Bruijn graph are monochromatic (i.e., all k-mers in a unitig have the same set of references of origin, or color). Specifically, the unitigs are kept in the dictionary in color order, thereby allowing for the encoding of the map from k-mers to their colors in as little as 1 + o(1) bits per unitig. Hence, one color per unitig is stored in the index with almost no space/time overhead. By combining this property with simple but effective compression methods for integer lists, the index achieves very small space. We implement these methods in a tool called Fulgor, and conduct an extensive experimental analysis to demonstrate the improvement of our tool over previous solutions. For example, compared to Themisto—the strongest competitor in terms of index space vs. query time trade-off—Fulgor requires significantly less space (up to 43% less space for a collection of 150,000 Salmonella enterica genomes), is at least twice as fast for color queries, and is 2-6x faster to construct

Fulgor: A Fast and Compact {k-mer} Index for Large-Scale Matching and Color Queries

The problem of sequence identification or matching - determining the subset of reference sequences from a given collection that are likely to contain a short, queried nucleotide sequence - is relevant for many important tasks in Computational Biology, such as metagenomics and pan-genome analysis. Due to the complex nature of such analyses and the large scale of the reference collections a resource-efficient solution to this problem is of utmost importance. This poses the threefold challenge of representing the reference collection with a data structure that is efficient to query, has light memory usage, and scales well to large collections. To solve this problem, we describe how recent advancements in associative, order-preserving, k-mer dictionaries can be combined with a compressed inverted index to implement a fast and compact colored de Bruijn graph data structure. This index takes full advantage of the fact that unitigs in the colored de Bruijn graph are monochromatic (all k-mers in a unitig have the same set of references of origin, or "color"), leveraging the order-preserving property of its dictionary. In fact, k-mers are kept in unitig order by the dictionary, thereby allowing for the encoding of the map from k-mers to their inverted lists in as little as 1+o(1) bits per unitig. Hence, one inverted list per unitig is stored in the index with almost no space/time overhead. By combining this property with simple but effective compression methods for inverted lists, the index achieves very small space. We implement these methods in a tool called Fulgor. Compared to Themisto, the prior state of the art, Fulgor indexes a heterogeneous collection of 30,691 bacterial genomes in 3.8× less space, a collection of 150,000 Salmonella enterica genomes in approximately 2× less space, is at least twice as fast for color queries, and is 2-6 × faster to construct

Swimming downstream: statistical analysis of differential transcript usage following Salmon quantification [version 2; referees: 1 approved, 2 approved with reservations]

Detection of differential transcript usage (DTU) from RNA-seq data is an important bioinformatic analysis that complements differential gene expression analysis. Here we present a simple workflow using a set of existing R/Bioconductor packages for analysis of DTU. We show how these packages can be used downstream of RNA-seq quantification using the Salmon software package. The entire pipeline is fast, benefiting from inference steps by Salmon to quantify expression at the transcript level. The workflow includes live, runnable code chunks for analysis using DRIMSeq and DEXSeq, as well as for performing two-stage testing of DTU using the stageR package, a statistical framework to screen at the gene level and then confirm which transcripts within the significant genes show evidence of DTU. We evaluate these packages and other related packages on a simulated dataset with parameters estimated from real data

A junction coverage compatibility score to quantify the reliability of transcript abundance estimates and annotation catalogs

Most methods for statistical analysis of RNA-seq data take a matrix of abundance estimates for some type of genomic features as their input, and consequently the quality of any obtained results is directly dependent on the quality of these abundances. Here, we present the junction coverage compatibility score, which provides a way to evaluate the reliability of transcript-level abundance estimates and the accuracy of transcript annotation catalogs. It works by comparing the observed number of reads spanning each annotated splice junction in a genomic region to the predicted number of junction-spanning reads, inferred from the estimated transcript abundances and the genomic coordinates of the corresponding annotated transcripts. We show that although most genes show good agreement between the observed and predicted junction coverages, there is a small set of genes that do not. Genes with poor agreement are found regardless of the method used to estimate transcript abundances, and the corresponding transcript abundances should be treated with care in any downstream analyses