An incrementally updatable and scalable system for large-scale sequence search using the Bentley-Saxe transformation.

MOTIVATION: In the past few years, researchers have proposed numerous indexing schemes for searching large datasets of raw sequencing experiments. Most of these proposed indexes are approximate (i.e. with one-sided errors) in order to save space. Recently, researchers have published exact indexes-Mantis, VariMerge and Bifrost-that can serve as colored de Bruijn graph representations in addition to serving as k-mer indexes. This new type of index is promising because it has the potential to support more complex analyses than simple searches. However, in order to be useful as indexes for large and growing repositories of raw sequencing data, they must scale to thousands of experiments and support efficient insertion of new data. RESULTS: In this paper, we show how to build a scalable and updatable exact raw sequence-search index. Specifically, we extend Mantis using the Bentley-Saxe transformation to support efficient updates, called Dynamic Mantis. We demonstrate Dynamic Mantis's scalability by constructing an index of ≈40K samples from SRA by adding samples one at a time to an initial index of 10K samples. Compared to VariMerge and Bifrost, Dynamic Mantis is more efficient in terms of index-construction time and memory, query time and memory and index size. In our benchmarks, VariMerge and Bifrost scaled to only 5K and 80 samples, respectively, while Dynamic Mantis scaled to more than 39K samples. Queries were over 24× faster in Mantis than in Bifrost (VariMerge does not immediately support general search queries we require). Dynamic Mantis indexes were about 2.5× smaller than Bifrost's indexes and about half as big as VariMerge's indexes. AVAILABILITY AND IMPLEMENTATION: Dynamic Mantis implementation is available at https://github.com/splatlab/mantis/tree/mergeMSTs. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

AGAMEMNON: an Accurate metaGenomics And MEtatranscriptoMics quaNtificatiON analysis suite.

We introduce AGAMEMNON ( https://github.com/ivlachos/agamemnon ) for the acquisition of microbial abundances from shotgun metagenomics and metatranscriptomic samples, single-microbe sequencing experiments, or sequenced host samples. AGAMEMNON delivers accurate abundances at genus, species, and strain resolution. It incorporates a time and space-efficient indexing scheme for fast pattern matching, enabling indexing and analysis of vast datasets with widely available computational resources. Host-specific modules provide exceptional accuracy for microbial abundance quantification from tissue RNA/DNA sequencing, enabling the expansion of experiments lacking metagenomic/metatranscriptomic analyses. AGAMEMNON provides an R-Shiny application, permitting performance of investigations and visualizations from a graphics interface.

PuffAligner: a fast, efficient and accurate aligner based on the Pufferfish index.

MOTIVATION: Sequence alignment is one of the first steps in many modern genomic analyses, such as variant detection, transcript abundance estimation and metagenomic profiling. Unfortunately, it is often a computationally expensive procedure. As the quantity of data and wealth of different assays and applications continue to grow, the need for accurate and fast alignment tools that scale to large collections of reference sequences persists. RESULTS: In this article, we introduce PuffAligner, a fast, accurate and versatile aligner built on top of the Pufferfish index. PuffAligner is able to produce highly sensitive alignments, similar to those of Bowtie2, but much more quickly. While exhibiting similar speed to the ultrafast STAR aligner, PuffAligner requires considerably less memory to construct its index and align reads. PuffAligner strikes a desirable balance with respect to the time, space and accuracy tradeoffs made by different alignment tools and provides a promising foundation on which to test new alignment ideas over large collections of sequences. AVAILABILITY AND IMPLEMENTATION: All the data used for preparing the results of this paper can be found with 10.5281/zenodo.4902332. PuffAligner is a free and open-source software. It is implemented in C++14 and can be obtained from https://github.com/COMBINE-lab/pufferfish/tree/cigar-strings. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Alignment and mapping methodology influence transcript abundance estimation.

BACKGROUND: The accuracy of transcript quantification using RNA-seq data depends on many factors, such as the choice of alignment or mapping method and the quantification model being adopted. While the choice of quantification model has been shown to be important, considerably less attention has been given to comparing the effect of various read alignment approaches on quantification accuracy. RESULTS: We investigate the influence of mapping and alignment on the accuracy of transcript quantification in both simulated and experimental data, as well as the effect on subsequent differential expression analysis. We observe that, even when the quantification model itself is held fixed, the effect of choosing a different alignment methodology, or aligning reads using different parameters, on quantification estimates can sometimes be large and can affect downstream differential expression analyses as well. These effects can go unnoticed when assessment is focused too heavily on simulated data, where the alignment task is often simpler than in experimentally acquired samples. We also introduce a new alignment methodology, called selective alignment, to overcome the shortcomings of lightweight approaches without incurring the computational cost of traditional alignment. CONCLUSION: We observe that, on experimental datasets, the performance of lightweight mapping and alignment-based approaches varies significantly, and highlight some of the underlying factors. We show this variation both in terms of quantification and downstream differential expression analysis. In all comparisons, we also show the improved performance of our proposed selective alignment method and suggest best practices for performing RNA-seq quantification.

An Efficient, Scalable, and Exact Representation of High-Dimensional Color Information Enabled Using de Bruijn Graph Search.

The colored de Bruijn graph (cdbg) and its variants have become an important combinatorial structure used in numerous areas in genomics, such as population-level variation detection in metagenomic samples, large-scale sequence search, and cdbg-based reference sequence indices. As samples or genomes are added to the cdbg, the color information comes to dominate the space required to represent this data structure. In this article, we show how to represent the color information efficiently by adopting a hierarchical encoding that exploits correlations among color classes-patterns of color occurrence-present in the de Bruijn graph (dbg). A major challenge in deriving an efficient encoding of the color information that takes advantage of such correlations is determining which color classes are close to each other in the high-dimensional space of possible color patterns. We demonstrate that the dbg itself can be used as an efficient mechanism to search for approximate nearest neighbors in this space. While our approach reduces the encoding size of the color information even for relatively small cdbgs (hundreds of experiments), the gains are particularly consequential as the number of potential colors (i.e., samples or references) grows into thousands. We apply this encoding in the context of two different applications; the implicit cdbg used for a large-scale sequence search index, Mantis, as well as the encoding of color information used in population-level variation detection by tools such as Vari and Rainbowfish. Our results show significant improvements in the overall size and scalability of representation of the color information. In our experiment on 10,000 samples, we achieved >11 × better compression compared to Ramen, Ramen, Rao (RRR).

A space and time-efficient index for the compacted colored de Bruijn graph.

Motivation: Indexing reference sequences for search-both individual genomes and collections of genomes-is an important building block for many sequence analysis tasks. Much work has been dedicated to developing full-text indices for genomic sequences, based on data structures such as the suffix array, the BWT and the FM-index. However, the de Bruijn graph, commonly used for sequence assembly, has recently been gaining attention as an indexing data structure, due to its natural ability to represent multiple references using a graphical structure, and to collapse highly-repetitive sequence regions. Yet, much less attention has been given as to how to best index such a structure, such that queries can be performed efficiently and memory usage remains practical as the size and number of reference sequences being indexed grows large. Results: We present a novel data structure for representing and indexing the compacted colored de Bruijn graph, which allows for efficient pattern matching and retrieval of the reference information associated with each k-mer. As the popularity of the de Bruijn graph as an index has increased over the past few years, so have the number of proposed representations of this structure. Existing structures typically fall into two categories; those that are hashing-based and provide very fast access to the underlying k-mer information, and those that are space-frugal and provide asymptotically efficient but practically slower pattern search. Our representation achieves a compromise between these two extremes. By building upon minimum perfect hashing and making use of succinct representations where applicable, our data structure provides practically fast lookup while greatly reducing the space compared to traditional hashing-based implementations. Further, we describe a sampling scheme for this index, which provides the ability to trade off query speed for a reduction in the index size. We believe this representation strikes a desirable balance between speed and space usage, and allows for fast search on large reference sequences. Finally, we describe an application of this index to the taxonomic read assignment problem. We show that by adopting, essentially, the approach of Kraken, but replacing k-mer presence with coverage by chains of consistent unique maximal matches, we can improve the space, speed and accuracy of taxonomic read assignment. Availability and implementation: pufferfish is written in C++11, is open source, and is available at https://github.com/COMBINE-lab/pufferfish. Supplementary information: Supplementary data are available at Bioinformatics online.

Mantis: A Fast, Small, and Exact Large-Scale Sequence-Search Index.

Sequence-level searches on large collections of RNA sequencing experiments, such as the NCBI Sequence Read Archive (SRA), would enable one to ask many questions about the expression or variation of a given transcript in a population. Existing approaches, such as the sequence Bloom tree, suffer from fundamental limitations of the Bloom filter, resulting in slow build and query times, less-than-optimal space usage, and potentially large numbers of false-positives. This paper introduces Mantis, a space-efficient system that uses new data structures to index thousands of raw-read experiments and facilitates large-scale sequence searches. In our evaluation, index construction with Mantis is 6× faster and yields a 20% smaller index than the state-of-the-art split sequence Bloom tree (SSBT). For queries, Mantis is 6-108× faster than SSBT and has no false-positives or -negatives. For example, Mantis was able to search for all 200,400 known human transcripts in an index of 2,652 RNA sequencing experiments in 82 min; SSBT took close to 4 days.

Grouper: graph-based clustering and annotation for improved de novo transcriptome analysis.

Motivation: De novo transcriptome analysis using RNA-seq offers a promising means to study gene expression in non-model organisms. Yet, the difficulty of transcriptome assembly means that the contigs provided by the assembler often represent a fractured and incomplete view of the transcriptome, complicating downstream analysis. We introduce Grouper, a new method for clustering contigs from de novo assemblies that are likely to belong to the same transcripts and genes; these groups can subsequently be analyzed more robustly. When provided with access to the genome of a related organism, Grouper can transfer annotations to the de novo assembly, further improving the clustering. Results: On de novo assemblies from four different species, we show that Grouper is able to accurately cluster a larger number of contigs than the existing state-of-the-art method. The Grouper pipeline is able to map greater than 10% more reads against the contigs, leading to accurate downstream differential expression analyses. The labeling module, in the presence of a closely related annotated genome, can efficiently transfer annotations to the contigs and use this information to further improve clustering. Overall, Grouper provides a complete and efficient pipeline for processing de novo transcriptomic assemblies. Availability and implementation: The Grouper software is freely available at https://github.com/COMBINE-lab/grouper under the 2-clause BSD license. Supplementary information: Supplementary data are available at Bioinformatics online.

Improved data-driven likelihood factorizations for transcript abundance estimation.

MOTIVATION: Many methods for transcript-level abundance estimation reduce the computational burden associated with the iterative algorithms they use by adopting an approximate factorization of the likelihood function they optimize. This leads to considerably faster convergence of the optimization procedure, since each round of e.g. the EM algorithm, can execute much more quickly. However, these approximate factorizations of the likelihood function simplify calculations at the expense of discarding certain information that can be useful for accurate transcript abundance estimation. RESULTS: We demonstrate that model simplifications (i.e. factorizations of the likelihood function) adopted by certain abundance estimation methods can lead to a diminished ability to accurately estimate the abundances of highly related transcripts. In particular, considering factorizations based on transcript-fragment compatibility alone can result in a loss of accuracy compared to the per-fragment, unsimplified model. However, we show that such shortcomings are not an inherent limitation of approximately factorizing the underlying likelihood function. By considering the appropriate conditional fragment probabilities, and adopting improved, data-driven factorizations of this likelihood, we demonstrate that such approaches can achieve accuracy nearly indistinguishable from methods that consider the complete (i.e. per-fragment) likelihood, while retaining the computational efficiently of the compatibility-based factorizations. AVAILABILITY AND IMPLEMENTATION: Our data-driven factorizations are incorporated into a branch of the Salmon transcript quantification tool: https://github.com/COMBINE-lab/salmon/tree/factorizations . CONTACT: rob.patro@cs.stonybrook.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.