4,169 research outputs found
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Deconvolute individual genomes from metagenome sequences through short read clustering.
Metagenome assembly from short next-generation sequencing data is a challenging process due to its large scale and computational complexity. Clustering short reads by species before assembly offers a unique opportunity for parallel downstream assembly of genomes with individualized optimization. However, current read clustering methods suffer either false negative (under-clustering) or false positive (over-clustering) problems. Here we extended our previous read clustering software, SpaRC, by exploiting statistics derived from multiple samples in a dataset to reduce the under-clustering problem. Using synthetic and real-world datasets we demonstrated that this method has the potential to cluster almost all of the short reads from genomes with sufficient sequencing coverage. The improved read clustering in turn leads to improved downstream genome assembly quality
Recovering complete and draft population genomes from metagenome datasets.
Assembly of metagenomic sequence data into microbial genomes is of fundamental value to improving our understanding of microbial ecology and metabolism by elucidating the functional potential of hard-to-culture microorganisms. Here, we provide a synthesis of available methods to bin metagenomic contigs into species-level groups and highlight how genetic diversity, sequencing depth, and coverage influence binning success. Despite the computational cost on application to deeply sequenced complex metagenomes (e.g., soil), covarying patterns of contig coverage across multiple datasets significantly improves the binning process. We also discuss and compare current genome validation methods and reveal how these methods tackle the problem of chimeric genome bins i.e., sequences from multiple species. Finally, we explore how population genome assembly can be used to uncover biogeographic trends and to characterize the effect of in situ functional constraints on the genome-wide evolution
Assembling large, complex environmental metagenomes
The large volumes of sequencing data required to sample complex environments
deeply pose new challenges to sequence analysis approaches. De novo metagenomic
assembly effectively reduces the total amount of data to be analyzed but
requires significant computational resources. We apply two pre-assembly
filtering approaches, digital normalization and partitioning, to make large
metagenome assemblies more comput\ ationaly tractable. Using a human gut mock
community dataset, we demonstrate that these methods result in assemblies
nearly identical to assemblies from unprocessed data. We then assemble two
large soil metagenomes from matched Iowa corn and native prairie soils. The
predicted functional content and phylogenetic origin of the assembled contigs
indicate significant taxonomic differences despite similar function. The
assembly strategies presented are generic and can be extended to any
metagenome; full source code is freely available under a BSD license.Comment: Includes supporting informatio
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Optimizing sequencing protocols for leaderboard metagenomics by combining long and short reads.
As metagenomic studies move to increasing numbers of samples, communities like the human gut may benefit more from the assembly of abundant microbes in many samples, rather than the exhaustive assembly of fewer samples. We term this approach leaderboard metagenome sequencing. To explore protocol optimization for leaderboard metagenomics in real samples, we introduce a benchmark of library prep and sequencing using internal references generated by synthetic long-read technology, allowing us to evaluate high-throughput library preparation methods against gold-standard reference genomes derived from the samples themselves. We introduce a low-cost protocol for high-throughput library preparation and sequencing
Extreme Scale De Novo Metagenome Assembly
Metagenome assembly is the process of transforming a set of short,
overlapping, and potentially erroneous DNA segments from environmental samples
into the accurate representation of the underlying microbiomes's genomes.
State-of-the-art tools require big shared memory machines and cannot handle
contemporary metagenome datasets that exceed Terabytes in size. In this paper,
we introduce the MetaHipMer pipeline, a high-quality and high-performance
metagenome assembler that employs an iterative de Bruijn graph approach.
MetaHipMer leverages a specialized scaffolding algorithm that produces long
scaffolds and accommodates the idiosyncrasies of metagenomes. MetaHipMer is
end-to-end parallelized using the Unified Parallel C language and therefore can
run seamlessly on shared and distributed-memory systems. Experimental results
show that MetaHipMer matches or outperforms the state-of-the-art tools in terms
of accuracy. Moreover, MetaHipMer scales efficiently to large concurrencies and
is able to assemble previously intractable grand challenge metagenomes. We
demonstrate the unprecedented capability of MetaHipMer by computing the first
full assembly of the Twitchell Wetlands dataset, consisting of 7.5 billion
reads - size 2.6 TBytes.Comment: Accepted to SC1
These are not the k-mers you are looking for: efficient online k-mer counting using a probabilistic data structure
K-mer abundance analysis is widely used for many purposes in nucleotide
sequence analysis, including data preprocessing for de novo assembly, repeat
detection, and sequencing coverage estimation. We present the khmer software
package for fast and memory efficient online counting of k-mers in sequencing
data sets. Unlike previous methods based on data structures such as hash
tables, suffix arrays, and trie structures, khmer relies entirely on a simple
probabilistic data structure, a Count-Min Sketch. The Count-Min Sketch permits
online updating and retrieval of k-mer counts in memory which is necessary to
support online k-mer analysis algorithms. On sparse data sets this data
structure is considerably more memory efficient than any exact data structure.
In exchange, the use of a Count-Min Sketch introduces a systematic overcount
for k-mers; moreover, only the counts, and not the k-mers, are stored. Here we
analyze the speed, the memory usage, and the miscount rate of khmer for
generating k-mer frequency distributions and retrieving k-mer counts for
individual k-mers. We also compare the performance of khmer to several other
k-mer counting packages, including Tallymer, Jellyfish, BFCounter, DSK, KMC,
Turtle and KAnalyze. Finally, we examine the effectiveness of profiling
sequencing error, k-mer abundance trimming, and digital normalization of reads
in the context of high khmer false positive rates. khmer is implemented in C++
wrapped in a Python interface, offers a tested and robust API, and is freely
available under the BSD license at github.com/ged-lab/khmer
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