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Metagenomic analysis of 30,000-year-old microbes from Siberian permafrost

A metagenomic survey of microbial DNA extracted from 30,000-year-old permafrost, and genome assembly of a giant virus which was resurrected from the sample

Fig. 1: Extraction of permafrost sample

Permafrost is an ideal storage medium for DNA preservation

Permafrost is a thick layer of soil, lying beneath ice or other soil, which stays frozen throughout the year. Permafrost in Chukotka, Siberia, is known to have remained frozen for tens of thousands of years (Fig. 1). Permafrost is an ideal storage medium for DNA preservation. We obtained DNA from a 30,000-year-old Siberian permafrost sample which had never thawed, and performed metagenomic analysis using our WIMP workflow. WIMP performs real-time species identification, using kraken to determine the most likely placement of a sequence in the taxonomy tree, and to give each placement a classification score. The higher this score, the higher the confidence in the classification

Fig. 2 WIMP analysis of permafrost DNA a) fungi, archaea and viruses b) bacteria

Analysis of permafrost DNA offers snapshot of life 30,000 years ago

The WIMP workflow currently analyses bacteria, viruses and fungi together, and we found DNA from examples of each type of microorganism in the sample. For the sake of clarity, results for viruses and fungi (Fig. 2a) are shown separately from those for bacteria (Fig. 2b).

Intact amoeba cells have been observed in Siberian permafrost samples. Because giant DNA viruses have been observed within modern amoeba cells, collaborators at CNRS in Marseille suspected that there could be preserved giant amoeba-infecting viral particles trapped in Siberian permafrost.

Fig. 3 ONT-only assembly of Pithovirus sibericum

30,000-year-old virus resurrected from Siberian permafrost

Our collaborators discovered a viable amphora-shaped virus in the Siberian permafrost sample (Fig. 3a). The virus was found to infect Acanthamoeba, allowing it to be cultured in the laboratory and studied. It was given the  name Pithovirus sibericum. By viral standards, Pithovirus is enormous, 1.5 mm long, and has a large AT-rich genome, approximately 600 kb in length. We prepared a low-input library from the cultured virus, and sequenced this on the MinIONTM. We assembled the genome using Nanocorrect and the Celera Assembler, obtaining a single contig of 620 kb. Our assembly agrees well with the published assembly, as illustrated in the Mummer plot (Fig. 3b).

Fig. 4 Thawing permafrost may release viable pathogens

Viable pathogens may be released from melting permafrost

Pithovirus shares several features, including genome structure and replication cycle, with other large eukaryote-infecting viruses, raising the possibility that other viruses are also preserved in permafrost in a viable state. As a consequence, melting of permafrost, as a result of climate change, mining or drilling may release these potential pathogens. No living cellular organisms have yet been definitively obtained from samples of this age, but some pathogenic bacteria are known to be capable of survival under the low temperatures encountered in circumpolar regions. It is therefore advisable to perform metagenomic surveys more widely on permafrosts to document the pathogens present. 

Incorporating sequence capture into library preparation for MinION and PromethION

Hybrid sequence capture allows users to select thousands of loci of interest simultaneously prior to sequencing, making more efficient use of the sequencing run.

Fig. 1 Long read sequence capture a) workflow b) multiplexed sequence capture

Sequence capture uses complementary probes to enrich for targets of interest

Sequence capture is a technique which allows the enrichment of specific regions of interest from a genome. It is useful when:
i) the user is not interested in analysing the entire genome
ii) the genome is too large for the throughput of the sequencer
iii) the user wishes to save money and time on sequencing and analysis
iv) the regions are longer than can be amplified by PCR, or too many PCRs would be required.

Sequence capture is performed during library preparation by hybridising the library fragments to probes which are specific to the regions of interest (Fig. 1).

Fig. 2 Analysis report of sequence-capture data for the human exome

Resequencing analysis workflow for sequence-capture experiments

We have released an updated analysis workflow for sequence-capture experiments. To illustrate this, we captured the human exome using Agilent’s SureSelect Human All Exon V6 panel, and generated ~ 2.35 Gb of 1D sequence data from a MinION run, representing ~40x average coverage. We analysed the data using the resequencing analysis workflow (Fig. 2). Following basecalling, reads are mapped to the human exome reference sequence. Individual gene information is displayed, including coverage and read-accuracy distribution at that position. Future releases of this application will support the uploading of target regions, highlight known SNPs in the target regions and allow those SNPs to be displayed with a confidence value.

Fig. 3 Plots showing reads enriched for the BRCA1 gene and SNP-calling

Enrichment of Comprehensive Cancer panel genes, allowing detection of SNPs

We evaluated Agilent’s ClearSeq Comprehensive Cancer panel using DNA from two BRCA1 SNP-carriers with positive family history of breast cancer (NA13708 and NA13710, Corriell NIGMS Human Genetic Cell Repository). We performed sequence capture as described, sequenced the library and basecalled reads using the 1D workflow. We aligned all reads to the reference sequence from Agilent, using BWA with standard parameters, and visualised reads with Savant (Fig. 3a). Figs. 3b and 3c show alignments of reads from exon 16 and exon 13, respectively, of the BRCA1 gene. Sequencing errors are distributed randomly, allowing SNPs to be seen clearly in the data.

Fig. 4 Long reads mapping to exons 1–27 of NBPF1 after whole exome sequence capture

Long fragment capture protocol generates reads which can span multiple exons

We performed sequence capture on human genomic DNA, using Agilent’s SureSelect Human All Exon V6, using a randomly fragmented library which had been size-selected using the Blue Pippin automated gel fractionation system, leaving only fragments above 6 kb. The library was then amplified by PCR prior to performing hybrid capture. The library was sequenced on a single MinION run and the reads were mapped to the human genome (H19). Fig. 4 shows reads mapping to the neuroblastoma BPF1 gene, and it can be seen that multiple exons are spanned by many of the reads, meaning that both exon- and intron-specific variations can be detected and phased haplotypes can be obtained.

BWA and LAST have been tuned to work with nanopore reads

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nanopolish – nanopore sequence analysis and genome assembly software

Jared Simpson, University of Toronto

Release Date: 04-Sept-2015

A nanopore consensus algorithm using a signal-level hidden Markov model.

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The Mystery of the Pink Lake

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Determining Exon Connectivity by Nanopore Sequencing

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Sequence capture on the MinION

A presentation at London Calling 2015 by Daniel Fordham.

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INC-Seq: Accurate single molecule reads using nanopore sequencing

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Nanopore Sequencing as a Rapidly Deployable Ebola Outbreak Tool

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NanoOK: Multi-reference alignment analysis of nanopore sequencing data, quality and error profiles

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LINKS: Scalable, alignment-free scaffolding of draft genomes with long reads

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Scaffolding of a bacterial genome using MinION nanopore sequencing

Second generation sequencing has revolutionized genomic studies. However, most genomes contain repeated DNA elements that are longer than the read lengths achievable with typical sequencers, so the genomic order of several generated contigs cannot be easily resolved.

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Nanopore sequencing for detection of pharmacogenomic variants and haplotypes

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An epidemiological river metagenome based on MinION

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Genome assembly using Nanopore-guided long and error-free DNA reads

Long-read sequencing technologies were launched a few years ago, and in contrast with short-read sequencing technologies, they offered a promise of solving assembly problems for large and complex genomes. Moreover by providing long-range information, it could also solve haplotype phasing.

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Improved data analysis for the MinION nanopore sequencer

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Long read nanopore sequencing for detection of HLA and CYP2D6 variants and haplotypes

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MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island

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Poretools: a toolkit for analyzing nanopore sequence data

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