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High-resolution mapping of evolutionary trajectories in a phage.

ABSTRACT: Experimental evolution in rapidly reproducing viruses offers a robust means to infer substitution trajectories during evolution. But with conventional approaches, this inference is limited by how many individual genotypes can be sampled from the population at a time. Low-frequency changes are difficult to detect, potentially rendering early stages of adaptation unobservable. Here we circumvent this using short-read sequencing technology in a fine-grained analysis of polymorphism dynamics in the sentinel organism: a single-stranded DNA phage PhiX174. Nucleotide differences were educed from noise with binomial filtering methods that harnessed quality scores and separate data from brief phage amplifications. Remarkably, a significant degree of variation was observed in all samples including those grown in brief 2-h cultures. Sites previously reported as subject to high-frequency polymorphisms over a course of weeks exhibited monotonic increases in polymorphism frequency within hours in this study. Additionally, even with limitations imposed by the short length of sequencing reads, we were able to observe statistically significant linkage among polymorphic sites in evolved lineages. Additional parallels between replicate lineages were apparent in the sharing of polymorphic sites and in correlated polymorphism frequencies. Missense mutations were more likely to occur than silent mutations. This study offers the first glimpse into "real-time" substitution dynamics and offers a robust conceptual framework for future viral resequencing studies.

SUBMITTER: Dickins B

PROVIDER: S-EPMC2817424 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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High-resolution mapping of evolutionary trajectories in a phage.

Dickins Benjamin B Nekrutenko Anton A

Genome biology and evolution 20090811

Experimental evolution in rapidly reproducing viruses offers a robust means to infer substitution trajectories during evolution. But with conventional approaches, this inference is limited by how many individual genotypes can be sampled from the population at a time. Low-frequency changes are difficult to detect, potentially rendering early stages of adaptation unobservable. Here we circumvent this using short-read sequencing technology in a fine-grained analysis of polymorphism dynamics in the ...[more]

PMID: 20333199

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Project description:Paraneoplastic neurological disorders are immune-mediated diseases understood to manifest as part of a misdirected anti-tumor immune response. Paraneoplastic neurological disorder-associated autoantibodies can assist with diagnosis and enhance our understanding of tumor-associated immune processes. We designed a comprehensive library of 49-amino-acid overlapping peptides spanning the entire human proteome, including all splicing isoforms and computationally predicted coding regions. Using this library, we optimized a phage immunoprecipitation and sequencing protocol with multiple rounds of enrichment to create high-resolution epitope profiles in serum and cerebrospinal fluid (CSF) samples from patients suffering from two common paraneoplastic neurological disorders, the anti-Yo (n = 36 patients) and anti-Hu (n = 44 patients) syndromes. All (100%) anti-Yo patient samples yielded enrichment of peptides from the canonical anti-Yo (CDR2 and CDR2L) antigens, while 38% of anti-Hu patients enriched peptides deriving from the nELAVL (neuronal embryonic lethal abnormal vision like) family of proteins, the anti-Hu autoantigenic target. Among the anti-Hu patient samples that were positive for nELAVL, we noted a restricted region of immunoreactivity. To achieve single amino acid resolution, we designed a novel deep mutational scanning phage library encoding all possible single-point mutants targeting the reactive nELAVL region. This analysis revealed a distinct preference for the degenerate motif, RLDxLL, shared by ELAVL2, 3 and 4. Lastly, phage immunoprecipitation sequencing identified several known autoantigens in these same patient samples, including peptides deriving from the cancer-associated antigens ZIC and SOX families of transcription factors. Overall, this optimized phage immunoprecipitation sequencing library and protocol yielded the high-resolution epitope mapping of the autoantigens targeted in anti-Yo and anti-Hu encephalitis patients to date. The results presented here further demonstrate the utility and high-resolution capability of phage immunoprecipitation sequencing for both basic science and clinical applications and for better understanding the antigenic targets and triggers of paraneoplastic neurological disorders.

Dataset Information

High-resolution mapping of evolutionary trajectories in a phage.

Publications

High-resolution mapping of evolutionary trajectories in a phage.

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