Jens Stoye

Phylogenetic classification of short environmental DNA fragments

Metagenomics is providing striking insights into the ecology of microbial communities. The recently developed massively parallel 454 pyrosequencing technique gives the opportunity to rapidly obtain metagenomic sequences at a low cost and without cloning bias. However, the phylogenetic analysis of the short reads produced represents a significant computational challenge. The phylogenetic algorithm CARMA for predicting the source organisms of environmental 454 reads is described. The algorithm searches for conserved Pfam domain and protein families in the unassembled reads of a sample. These gene fragments (environmental gene tags, EGTs), are classified into a higher-order taxonomy based on the reconstruction of a phylogenetic tree of each matching Pfam family. The method exhibits high accuracy for a wide range of taxonomic groups, and EGTs as short as 27 amino acids can be phylogenetically classified up to the rank of genus. The algorithm was applied in a comparative study of three aquatic microbial samples obtained by 454 pyrosequencing. Profound differences in the taxonomic composition of these samples could be clearly revealed.

A unifying view of genome rearrangements

Genome rearrangements have been modeled by a variety of operations such as inversions, translocations, fissions, fusions, transpositions and block interchanges. The double cut and join operation, introduced by Yancopoulos et al., allows to model all the classical operations while simplifying the algorithms. In this paper we show a simple way to apply this operation to the most general type of genomes with a mixed collection of linear and circular chromosomes. We also describe a graph structure that allows simplifying the theory and distance computation considerably, as neither capping nor concatenation of the linear chromosomes are necessary.

Taxonomic composition and gene content of a methane-producing microbial community isolated from a biogas reactor.

A total community DNA sample from an agricultural biogas reactor continuously fed with maize silage, green rye, and small proportions of chicken manure has recently been sequenced using massively parallel pyrosequencing. In this study, the sample was computationally characterized without a prior assembly step, providing quantitative insights into the taxonomic composition and gene content of the underlying microbial community. Clostridiales from the phylum Firmicutes is the most prevalent phylogenetic order, Methanomicrobiales are dominant among methanogenic archaea. An analysis of Operational Taxonomic Units (OTUs) revealed that the entire microbial community is only partially covered by the sequenced sample, despite that estimates suggest only a moderate overall diversity of the community. Furthermore, the results strongly indicate that archaea related to the genus Methanoculleus, using CO2 as electron acceptor and H2 as electron donor, are the main producers of methane in the analyzed biogas reactor sample. A phylogenetic analysis of glycosyl hydrolase protein families suggests that Clostridia play an important role in the digestion of polysaccharides and oligosaccharides. Finally, the results unveiled that most of the organisms constituting the sample are still unexplored.

Updating benchtop sequencing performance comparison

In April 2012, your journal published a study by Loman et al.1 that systematically compared desktop next-generation sequencers (NGS) from three instrument providers. Using the custom scripts supplied by the authors, the same software and the same draft genome (with 153 remaining gaps within several scaffolds) as the reference, we reproduced their results with their data of the enterohemorrhagic Escherichia coli (EHEC) strain found in the 2011 outbreak in Germany. However, we wish to bring readers’ attention to some shortcomings in the report from Loman et al.1, focusing particularly on its discussion of read-level error analysis. NGS is a rapidly changing market, which clearly complicates the comparisons such as that made by Loman et al. Since the original study1, Illumina (San Diego) has launched the MiSeq sequencer officially and has released Nextera library construction kits and 2 × 250–base-pair (250-bp) paired-end (PE) sequencing chemistry. Furthermore, Life Technologies (Carlsbad, California), has made 200-bp and 300-bp kits available for the Ion Torrent Personal Genome Machine (PGM). Roche (Basel, Switzerland) has updated the Sequencing System software for its 454 GS Junior (GSJ) from version 2.6 to 2.7. In this report, we provide an up-to-date snapshot of how benchtop platforms have evolved since the previous study1.

Linear time algorithms for finding and representing all the tandem repeats in a string

A tandem repeat (or square) is a string αα, where α is a non-empty string. We present an O(|S|)-time algorithm that operates on the suffix tree T(S) for a string S, finding and marking the endpoint in T(S) of every tandem repeat that occurs in S. This decorated suffix tree implicitly represents all occurrences of tandem repeats in S, and can be used to efficiently solve many questions concerning tandem repeats and tandem arrays in S. This improves and generalizes several prior efforts to efficiently capture large subsets of tandem repeats.

Efficient implementation of lazy suffix trees

We present an efficient implementation of a write‐only top‐down construction for suffix trees. Our implementation is based on a new, space‐efficient representation of suffix trees that requires only 12 bytes per input character in the worst case, and 8.5 bytes per input character on average for a collection of files of different type. We show how to efficiently implement the lazy evaluation of suffix trees such that a subtree is evaluated only when it is traversed for the first time. Our experiments show that for the problem of searching many exact patterns in a fixed input string, the lazy top‐down construction is often faster and more space efficient than other methods. Copyright

Efficient q -gram filters for finding all ε-matches over a given length

Fast and exact comparison of large genomic sequences remains a challenging task in biosequence analysis. We consider the problem of finding all epsilon-matches between two sequences, i.e., all local alignments over a given length with an error rate of at most epsilon. We study this problem theoretically, giving an efficient q-gram filter for solving it. Two applications of the filter are also discussed, in particular genomic sequence assembly and BLAST-like sequence comparison. Our results show that the method is 25 times faster than BLAST, while not being heuristic.

Finding All Common Intervals of k Permutations

Given k permutations of n elements, a k-tuple of intervals of these permutations consisting of the same set of elements is called a common interval. We present an algorithm that finds in a family of k permutations of n elements all K common intervals in optimal O(nk+K) time and O(n) additional space.This extends a result by Uno and Yagiura (Algorithmica 26, 290-309, 2000) who present an algorithm to find all K common intervals of k = 2 permutations in optimal O(n+K) time and O(n) space. To achieve our result, we introduce the set of irreducible intervals, a generating subset of the set of all common intervals of k permutations.

r2cat: synteny plots and comparative assembly

Summary: Recent parallel pyrosequencing methods and the increasing number of finished genomes encourage the sequencing and investigation of closely related strains. Although the sequencing itself becomes easier and cheaper with each machine generation, the finishing of the genomes remains difficult. Instead of the desired whole genomic sequence, a set of contigs is the result of the assembly. In this applications note, we present the tool r2cat (related reference contig arrangement tool) that helps in the task of comparative assembly and also provides an interactive visualization for synteny inspection. Availability: http://bibiserv.techfak.uni-bielefeld.de/r2cat Contact: peter.husemann@cebitec.uni-bielefeld.de

Bacterial Community Shift in Treated Periodontitis Patients Revealed by Ion Torrent 16S rRNA Gene Amplicon Sequencing

Periodontitis, one of the most common diseases in the world, is caused by a mixture of pathogenic bacteria and inflammatory host responses and often treated by antimicrobials as an adjunct to scaling and root planing (SRP). Our study aims to elucidate explorative and descriptive temporal shifts in bacterial communities between patients treated by SRP alone versus SRP plus antibiotics. This is the first metagenomic study using an Ion Torrent Personal Genome Machine (PGM). Eight subgingival plaque samples from four patients with chronic periodontitis, taken before and two months after intervention were analyzed. Amplicons from the V6 hypervariable region of the 16S rRNA gene were generated and sequenced each on a 314 chip. Sequencing reads were clustered into operational taxonomic units (OTUs, 3% distance), described by community metrics, and taxonomically classified. Reads ranging from 599,933 to 650,416 per sample were clustered into 1,648 to 2,659 non-singleton OTUs, respectively. Increased diversity (Shannon and Simpson) in all samples after therapy was observed regardless of the treatment type whereas richness (ACE) showed no correlation. Taxonomic analysis revealed different microbial shifts between both therapy approaches at all taxonomic levels. Most remarkably, the genera Porphyromonas, Tannerella, Treponema, and Filifactor all harboring periodontal pathogenic species were removed almost only in the group treated with SPR and antibiotics. For the species T. forsythia and P. gingivalis results were corroborated by real-time PCR analysis. In the future, hypothesis free metagenomic analysis could be the key in understanding polymicrobial diseases and be used for therapy monitoring. Therefore, as read length continues to increase and cost to decrease, rapid benchtop sequencers like the PGM might finally be used in routine diagnostic.