Alevin-fry unlocks rapid, accurate and memory-frugal quantification of single-cell RNA-seq data.

The rapid growth of high-throughput single-cell and single-nucleus RNA-sequencing (scRNA-seq and snRNA-seq) technologies has produced a wealth of data over the past few years. The size, volume and distinctive characteristics of these data necessitate the development of new computational methods to accurately and efficiently quantify sc/snRNA-seq data into count matrices that constitute the input to downstream analyses. We introduce the alevin-fry framework for quantifying sc/snRNA-seq data. In addition to being faster and more memory frugal than other accurate quantification approaches, alevin-fry ameliorates the memory scalability and false-positive expression issues that are exhibited by other lightweight tools. We demonstrate how alevin-fry can be effectively used to quantify sc/snRNA-seq data, and also how the spliced and unspliced molecule quantification required as input for RNA velocity analyses can be seamlessly extracted from the same preprocessed data used to generate normal gene expression count matrices.

Sigmoni: classification of nanopore signal with a compressed pangenome index.

SUMMARY: Improvements in nanopore sequencing necessitate efficient classification methods, including pre-filtering and adaptive sampling algorithms that enrich for reads of interest. Signal-based approaches circumvent the computational bottleneck of basecalling. But past methods for signal-based classification do not scale efficiently to large, repetitive references like pangenomes, limiting their utility to partial references or individual genomes. We introduce Sigmoni: a rapid, multiclass classification method based on the r-index that scales to references of hundreds of Gbps. Sigmoni quantizes nanopore signal into a discrete alphabet of picoamp ranges. It performs rapid, approximate matching using matching statistics, classifying reads based on distributions of picoamp matching statistics and co-linearity statistics, all in linear query time without the need for seed-chain-extend. Sigmoni is 10-100× faster than previous methods for adaptive sampling in host depletion experiments with improved accuracy, and can query reads against large microbial or human pangenomes. Sigmoni is the first signal-based tool to scale to a complete human genome and pangenome while remaining fast enough for adaptive sampling applications. AVAILABILITY AND IMPLEMENTATION: Sigmoni is implemented in Python, and is available open-source at https://github.com/vshiv18/sigmoni.

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.

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.

Movi: a fast and cache-efficient full-text pangenome index.

Efficient pangenome indexes are promising tools for many applications, including rapid classification of nanopore sequencing reads. Recently, a compressed-index data structure called the "move structure" was proposed as an alternative to other BWT-based indexes like the FM index and r-index. The move structure uniquely achieves both O(r) space and O(1)-time queries, where r is the number of runs in the pangenome BWT. We implemented Movi, an efficient tool for building and querying move-structure pangenome indexes. While the size of the Movi's index is larger than the r-index, it scales at a smaller rate for pangenome references, as its size is exactly proportional to r, the number of runs in the BWT of the reference. Movi can compute sophisticated matching queries needed for classification - such as pseudo-matching lengths and backward search - at least ten times faster than the fastest available methods, and in some cases more than 30-fold faster. Movi achieves this speed by leveraging the move structure's strong locality of reference, incurring close to the minimum possible number of cache misses for queries against large pangenomes. We achieve still further speed improvements by using memory prefetching to attain a degree of latency hiding that would be difficult with other index structures like the r-index. Movi's fast constant-time query loop makes it well suited to real-time applications like adaptive sampling for nanopore sequencing, where decisions must be made in a small and predictable time interval.

Sigmoni: classification of nanopore signal with a compressed pangenome index.

Improvements in nanopore sequencing necessitate efficient classification methods, including pre-filtering and adaptive sampling algorithms that enrich for reads of interest. Signal-based approaches circumvent the computational bottleneck of basecalling. But past methods for signal-based classification do not scale efficiently to large, repetitive references like pangenomes, limiting their utility to partial references or individual genomes. We introduce Sigmoni: a rapid, multiclass classification method based on the r-index that scales to references of hundreds of Gbps. Sigmoni quantizes nanopore signal into a discrete alphabet of picoamp ranges. It performs rapid, approximate matching using matching statistics, classifying reads based on distributions of picoamp matching statistics and co-linearity statistics. Sigmoni is 10-100× faster than previous methods for adaptive sampling in host depletion experiments with improved accuracy, and can query reads against large microbial or human pangenomes.

SEESAW: detecting isoform-level allelic imbalance accounting for inferential uncertainty.

Detecting allelic imbalance at the isoform level requires accounting for inferential uncertainty, caused by multi-mapping of RNA-seq reads. Our proposed method, SEESAW, uses Salmon and Swish to offer analysis at various levels of resolution, including gene, isoform, and aggregating isoforms to groups by transcription start site. The aggregation strategies strengthen the signal for transcripts with high uncertainty. The SEESAW suite of methods is shown to have higher power than other allelic imbalance methods when there is isoform-level allelic imbalance. We also introduce a new test for detecting imbalance that varies across a covariate, such as time.

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.

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.