We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 4

1.
Figure 1

Figure 1. Experimental protocol for assessing mutational capacity in different disease states. From: Use of whole genome sequencing to estimate the mutation rate of Mycobacterium tuberculosis during latent infection.

1) Cynomolgus macaques were infected with ~25CFU of Mtb Erdman via bronchoscopy. 2) Animals were euthanized in the indicated stages of disease for strain isolation. 3) 18 pathologic lesions were plated for bacterial colonies. 33 strains were isolated for WGS. 4) Genomic DNA was isolated from these strains and then analyzed via Illumina sequencing. 5) Reads were assembled using both de novo and scaffolded approaches. 15 SNPs were predicted by both methodologies. Insertions and deletions were not detected using either methodology. 6) Sanger sequencing confirmed 14 of the 15 putative SNPs identified by both scaffolded and de novo analysis.

Christopher B. Ford, et al. Nat Genet. ;43(5):482-486.
2.
Figure 4

Figure 4. Mutations in Mtb isolated from macaques with latent infection and related human isolates are putative products of oxidative damage. From: Use of whole genome sequencing to estimate the mutation rate of Mycobacterium tuberculosis during latent infection.

(a) Ten of the fourteen mutations observed in this study could be the product of oxidative damage: the deamination of cytosine (GC>AT) or the production of 7,8-dihydro-8-oxoguanine (GC>TA) by the oxidation of guanine. One of each type of mutation observed was seen in active disease (four mutations total). In contrast, eight of ten mutations observed in latent and reactivated disease are potential products of oxidative damage. There is a similar mutational spectra observed in the synonymous SNPs identified by WGS of a set of closely related strains from South Africa9. (b) These observations lead to a model of mutational pressures on Mtb during active disease and latent infection in which oxidative damage may play a central role in the generation of mutation.

Christopher B. Ford, et al. Nat Genet. ;43(5):482-486.
3.
Figure 2

Figure 2. WGS identifies SNPs in strains isolated from animals with active, latent, and reactivated latent infection. From: Use of whole genome sequencing to estimate the mutation rate of Mycobacterium tuberculosis during latent infection.

SNPs were predicted through WGS in 33 Mtb strains isolated from nine cynomolgus macaques at various stages of disease. All SNPs predicted through WGS were confirmed via Sanger sequencing or through independent identification by WGS. Genome coverage and the original notation used to describe each animal are found in Supplementary Table 1. The total length of infection in days is listed for each animal below the animal identifier (A–I). Lesion locations are abbreviated as follows: LLL – left lower lobe, RLL – right lower lobe, RML – right middle lobe, RUL – right upper lobe, ACL – accessory lobe, CN – cranial lymph node, BN – bronchial lymph node. Inoculum represents the sequence at the given coordinate of the inoculating strain, Mtb Erdman.

Christopher B. Ford, et al. Nat Genet. ;43(5):482-486.
4.
Figure 3

Figure 3. The mutational capacity of strains from latency and reactivated disease is similar to that of strains from active disease or in vitro growth. From: Use of whole genome sequencing to estimate the mutation rate of Mycobacterium tuberculosis during latent infection.

(a–c) Mutation rate (μ was estimated based on the number of unique SNPs (m) observed in each condition (4 active, 3 latent, 7 reactivated). This calculation was performed over a range of generation times (g, 18–240 hours per generation) to allow for the uncertainty in growth rate in vivo. The probability of observing μ when g is fixed at any given time was determined to build the probability distribution function around each estimate and to define the 95% confidence intervals. The single base mutation rate of the bacterium during in vitro growth (μin vitro) was determined by fluctuation analysis (Supplementary Figs. 1a–c) and is indicated by an arrow. In each clinical condition, μ20 (the predicted mutation rate if the generation time in vivo were as rapid as the generation time in vitro) is similar to μin vitro. Generation time in vivo is predicted to be substantially slower than in vitro, and thus the mutation rate must be proportionally higher to produce the observed number of SNPs. (d) Given the uncertainty in generation time, a mutation rate per day can be calculated to determine the rate at which mutations occur regardless of generation time. Mutations occur at a similar rate per day regardless of the disease status of the host. Error bars represent 95% confidence intervals.

Christopher B. Ford, et al. Nat Genet. ;43(5):482-486.

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk