MetaCAA: A clustering-aided methodology for efficient assembly of metagenomic datasets

Reddy, Rachamalla Maheedhar; Mohammed, Monzoorul Haque; Mande, Sharmila S.

doi:10.1016/j.ygeno.2014.02.007

Cited by 17 publications

(10 citation statements)

References 28 publications

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“…The retrospective nature of this study allowed us to compare the success of the strategies in retrieving three novel viral genomes from 454 metagenomic data. While different metagenome assembly strategies, especially for very large datasets of short read data, apply k-mer clustering or digital normalization and partitioning prior to assembly (Howe et al, 2014 ; Reddy et al, 2014 ), we here concentrated on strategies to link contigs after assembly in silico . Theoretically, these strategies can also be applied to contigs from metagenomes produced by other sequencing methods.…”

Section: Discussionmentioning

confidence: 99%

Assembly of viral genomes from metagenomes

Smits

Bodewes²,

Ruiz‐González

et al. 2014

Front. Microbiol.

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Viral infections remain a serious global health issue. Metagenomic approaches are increasingly used in the detection of novel viral pathogens but also to generate complete genomes of uncultivated viruses. In silico identification of complete viral genomes from sequence data would allow rapid phylogenetic characterization of these new viruses. Often, however, complete viral genomes are not recovered, but rather several distinct contigs derived from a single entity are, some of which have no sequence homology to any known proteins. De novo assembly of single viruses from a metagenome is challenging, not only because of the lack of a reference genome, but also because of intrapopulation variation and uneven or insufficient coverage. Here we explored different assembly algorithms, remote homology searches, genome-specific sequence motifs, k-mer frequency ranking, and coverage profile binning to detect and obtain viral target genomes from metagenomes. All methods were tested on 454-generated sequencing datasets containing three recently described RNA viruses with a relatively large genome which were divergent to previously known viruses from the viral families Rhabdoviridae and Coronaviridae. Depending on specific characteristics of the target virus and the metagenomic community, different assembly and in silico gap closure strategies were successful in obtaining near complete viral genomes.

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Section: Discussionmentioning

confidence: 99%

Assembly of viral genomes from metagenomes

Smits

Bodewes²,

Ruiz‐González

et al. 2014

Front. Microbiol.

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“…For these purposes we utilized the program CAP3 (58) that was originally developed for the assembly of large-insert clones sequenced using capillary sequencing, but has also been applied to de novo assembly of short read RNA-seq (77) and metagenomic sequence data (78). For each putative site, we retrieved any insertion-supporting read pairs as reported by RetroSeq and proximal soft-clipped reads, and then performed de novo assemblies with CAP3 ( Figure 1) run with parameters adjusted for joining smaller overlaps that could be expected from 101 bp reads (also see Methods).…”

Section: Precise Assembly Of Full-length Alu Variants Using Read Datamentioning

confidence: 99%

Discovery and characterization ofAlurepeat sequences via precise local read assembly

Wildschutte

Baron

Diroff

et al. 2015

Preprint

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Alu insertions have contributed to >11% of the human genome and ∼30-35 Alu subfamilies remain actively mobile, yet the characterization of polymorphic Alu insertions from short-read data remains a challenge. We build on existing computational methods to combine Alu detection and de novo assembly of WGS data as a means to reconstruct the full sequence of insertion events from Illumina paired end reads. Comparison with published calls obtained using PacBio long-reads indicates a false discovery rate below 5%, at the cost of reduced sensitivity due to the colocation of reference and non-reference repeats. We generate a highly accurate call set of 1614 completely assembled Alu variants from 53 samples from the Human Genome Diversity Project (HGDP) panel. We utilize the reconstructed alternative insertion haplotypes to genotype 1010 fully assembled insertions, obtaining >99% agreement with genotypes obtained by PCR. In our assembled sequences, we find evidence of premature insertion mechanisms and observe 5 truncation in 16% of AluYa5 and AluYb8 insertions. The sites of truncation coincide with stem-loop structures and SRP9/14 binding sites in the Alu RNA, implicating L1 ORF2p pausing in the generation of 5 truncations. Additionally, we identified variable AluJ and AluS elements that likely arose due to non-retrotransposition mechanisms.

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“…While recent efforts in short read assembly have focused on a de brujin graph approach ( 74 – 76 ), we reasoned a local assembly using an overlap-layout-consensus approach would take full advantage of our data. For these purposes we utilized the program CAP3 ( 58 ) that was originally developed for the assembly of large-insert clones sequenced using capillary sequencing, but has also been applied to de novo assembly of short read RNA-seq ( 77 ) and metagenomic sequence data ( 78 ). For each putative site, we retrieved any insertion-supporting read pairs as reported by RetroSeq and proximal soft-clipped reads, and then performed de novo assemblies with CAP3 (Figure 1 ) run with parameters adjusted for joining smaller overlaps that could be expected from 101 bp reads (also see Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

Discovery and characterization ofAlurepeat sequences via precise local read assembly

et al. 2015

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Alu insertions have contributed to >11% of the human genome and ∼30–35 Alu subfamilies remain actively mobile, yet the characterization of polymorphic Alu insertions from short-read data remains a challenge. We build on existing computational methods to combine Alu detection and de novo assembly of WGS data as a means to reconstruct the full sequence of insertion events from Illumina paired end reads. Comparison with published calls obtained using PacBio long-reads indicates a false discovery rate below 5%, at the cost of reduced sensitivity due to the colocation of reference and non-reference repeats. We generate a highly accurate call set of 1614 completely assembled Alu variants from 53 samples from the Human Genome Diversity Project (HGDP) panel. We utilize the reconstructed alternative insertion haplotypes to genotype 1010 fully assembled insertions, obtaining >99% agreement with genotypes obtained by PCR. In our assembled sequences, we find evidence of premature insertion mechanisms and observe 5′ truncation in 16% of AluYa5 and AluYb8 insertions. The sites of truncation coincide with stem-loop structures and SRP9/14 binding sites in the Alu RNA, implicating L1 ORF2p pausing in the generation of 5′ truncations. Additionally, we identified variable AluJ and AluS elements that likely arose due to non-retrotransposition mechanisms.

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MetaCAA: A clustering-aided methodology for efficient assembly of metagenomic datasets

Cited by 17 publications

References 28 publications

Assembly of viral genomes from metagenomes

Assembly of viral genomes from metagenomes

Discovery and characterization ofAlurepeat sequences via precise local read assembly

Discovery and characterization ofAlurepeat sequences via precise local read assembly

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