Products & Services
Applications

Applications

Nanopore sequencing offers advantages in all areas of research. Our offering includes DNA sequencing, as well as RNA and gene expression analysis and future technology for analysing proteins.

Learn about applications
View all Applications
Resources
News Explore
Contact
Resource Centre

MetaPore-C: using chromatin conformation capture and long nanopore reads for metagenomic analysis

Poster

Date: 3rd December 2020

Cross-linking can be used to fix all of the DNA in a cell, enabling more straightforward metagenomic assembly and allowing plasmids to be associated with their host genomes

Download the PDF

Fig. 1 MetaPore-C a) laboratory workflow b) overview of bioinformatics workflow c) correct association of plasmids with host genomes for a mixture of two bacterial species

Generating contact information from metagenomic mixtures, using chromatin conformation capture without restriction digestion or PCR, combined with nanopore sequencing

Chromatin conformation capture is a method used to investigate interactions between genomic loci that are not adjacent in the primary sequence. When applied to metagenomic mixtures, as well as simplifying assembly the technique provides a way to associate plasmids with their host genomes. It is typical to use restriction digestion to fragment cross-linked DNA before proximity ligation, but this step is influenced by the base composition of the genomes present, which is not always known in advance. Our protocol avoids digestion by using bead beating to simultaneously lyse the cells and to fragment the DNA (Fig. 1a). To produce contact information, MetaPore-C reads are first aligned to a collection of chromosomal and extra-chromosomal reference sequences using BWA-SW. Aligned reads are filtered to retain the minimal collection of alignments that traverse the majority of the read. The reference genomes are then divided into equally sized bins and each aligned segment of the MetaPore-C read is assigned a bin. Finally, the total number of bin-to-bin contacts is calculated from all reads and visualised in a contact map. Extra-chromosomal elements can be assigned to their host by determining which chromosome(s) share the most contacts with the element (Fig. 1b). This approach allowed us to assign plasmids to the correct host in a mixture of two bacteria (Fig. 1c).

Fig. 2 MetaPore-C of a probiotic sample showing host-plasmid interactions

Identifying intra- and extra-chromosomal contacts in a probiotic sample

We generated MetaPore-C data on a probiotic food supplement, which contained 15 known bacterial strains (Fig. 2a) and created contact maps for the bacterial chromosomes and plasmids within this sample (Figs. 2b and 2c). The plot of average nucleotide identity (Fig. 2d) reveals a low level of spurious interaction between species, most probably due to mapping ambiguities. Future work will involve the use of more fine-grained approaches to suppress this background signal. Figs. 2e and 2f summarise the contacts for each bacterial chromosome. Plasmids were associated to the expected host genomes and we identified intra-chromosomal interactions, which are valuable for binning and hence assembly.

Fig. 3 Associating plasmids with host genomes a) contact map b) proportion of interactions

Using MetaPore-C contacts to associate plasmids with their host genomes

To see if we could correctly associate plasmids even in extremely similar bacterial host genomes, we created a MetaPore-C contact map for a mock community with 6 bacterial strains and their plasmids (Fig. 3a). The community includes two E. coli strains, plus S. enterica, which shares >80% ANI with E. coli. The long regions of high contact density reveal the plasmid-genome contacts. There are small amounts of spurious, between-species interactions, due to ambiguous mapping between highly homologous regions. When the proportion of contacts is plotted for each possible plasmid-genome interaction, in every case the overwhelming majority of contacts is with the correct host genome (Fig. 3b).

Recommended for you

Open a chat to talk to our sales team
FAQs

FAQs

Search