Carroll, W.

Choo, L.

Chopra, A.

Clark, H.

Kermode, A.

Leary, S.

Nolan, D.

Strautins, K.

Tschochner, M.

English

Research Repository

MethodsTschochner M. 1, Leary S. 1, Strautins K. 1, Clark H. 1, Chopra A.1, Choo L. 1, Carroll W.2, Kermode A.2, Nolan D.1,31 Institute for Immunology & Infectious Diseases, Murdoch University, Perth, Western Australia3 Sir Charles Gairdner Hospital ,Nedlands, Perth, Western Australia 3 Department of Clinical Immunology & Immunogenetics, Royal Perth Hospital, Perth, Western Australia; Identifying Epstein-Barr virus EBNA-1 sequence variation using 454 FLX technology in Multiple Sclerosis patient samples AcknowledgmentsIntroductionMS samples were utilised from the Perth Demyelinating Disease Database (PDDD) (Western Australia). DNA was isolated from buffy coats and the C-terminal end of the EBNA-1 gene was amplified using a novel semi-nested PCR approach with a fully automated setup utilising Biomek FX robots. First round PCR was performed using Roche High Fidelity Taq in a 25µl reaction with primers EBV109111F-EBV109951R, resulting in a 840bp fragment. A semi-nested PCR was followed using the primer combination EBV109111F-EBV109869R resulting in a final 749 base pair product (Fig.1). Successful PCR samples were purified using AMPure and Sanger sequenced on an automated 96 capillary ABI 373 DNA sequencer, followed by analysis using the ASSIGN V4.0.1.36 software (Conexio Genomics) (Fig.2). Threshold for mixture detection in sanger sequencing has been established to be ~20%. To study sequence minorities in quasispecies present at >=1%, ultra-deepsequencing was performed using the FLX-454 Roche technology for asubset of 23 MS patients and a B95-8 control. The same first roundPCR products obtained were used for high throughput sequencing andSanger sequencing approaches. For the second round semi-nestedPCR, primers were 5′ end tagged with 10 nucleotides of uniquesequence (one each primer pair and sample) to enable pooling ofsamples in one library. Samples were purified using AMPure andconcentrations measured with Nanodrop. Copy number per samplewas calculated and 1x1011 copies per sample were pooled. Afterequimolar pooling, the combined samples were purified withminielute spin column (Qiagen). Final concentration for the pooledfragments was 150.4 ng/ul. One library of all pooled samples wasprepared and 454 sequenced (Fig.2).Correspondence: m.tschochner@iiid.com.auBoth genetic and environmental factors contribute to Multiple Sclerosis (MS)risk. Susceptibility to MS is conferred by immune system associated genes, withhuman leukocyte antigen (HLA) DRB1*1501 identified as a major MS risk allele.Infection with Epstein-Barr Virus (EBV) has also been shown to increase MS risk,and in particular anti-EBV nuclear antigen-1 (EBNA-1) antibodies are stronglyassociated with the disease. Additionally, it has been shown that EBNA1-specificCD4+ T-cells can cross-recognize MS-associated myelin antigens, and areselectively expanded in MS patients.We therefore sought to assess MS patient-specific EBNA-1 sequence variation,including the potential influence of minority sequence variants, in shapingimmune responses to known and predicted HLA-DRB1*1501-restrictedepitopes.Epitope coding sequence variability was directly compared using 14 samplessequenced with Sanger sequencing and 454 FLX technology for threepreviously described epitopes: a putative B cell epitope GRRPFFHPVGE(aa 401-411), and two HLA-DRB1*1501 epitopes (AEGLRALLARSHVER(aa 482-496), MVFLQTHIFAEVLKD (aa 563-477).As shown in Figure 5, both methods were concordant in detecting sequencevariation potentially relevant to epitope recognition and presentation,with only a minority of sequences sharing identity with the B95-8reference strain. 454 FLX technology identified mixtures of >1%,although generally at a low percentage with no mixtures > 10% withinthe epitopes.DiscussionThis study confirms the feasibility of direct amplification and sequencing ofEBNA-1 from patient samples in identifying patient-specific sequencevariation, with evidence of high level of concordance between sangerand FLX 454 sequencing techniques.The frequent detection of sequences that are divergent from B95-8 referencestrain suggests that studies investigating epitope-specific immuneresponses can be usefully guided by autologous patient sequenceinformation.Low-level sequence variation was identified by sensitive FLX technology but wasgenerally rare. It remains to be determined if these minority speciesaffect EBV-specific immune responses or disease outcomes.Amino acid variability was low and Sanger sequencing results revealed no mixedpositions. Utilizing 454 FLX deep sequencing, minor species could bedetected at >10% frequency in 2.9% (21 positions of all 714 positionsinvestigated), increasing to 25.5% of all positions investigated withminor species detected at >=1% (Table 2).At a 1% cut off, 105 positions contained mixed amino acids for one sample onlywhereas in 30 different positions there were at least 2 samples withan amino acid mixture in the same position (Table 2).14 samples tested on FLX had high coverage with average reads between 80-1632 for the C-terminal EBNA-1 fragment. 10 samples had low amountof coverage and or reads (average reads <15). Four of these samples(38585, 38695, 38939, 38593) were successful for PCR amplificationbut could not be sequenced with conventional Sanger sequencingtechnology. Unmatched contigs were blasted and were found torepresent chromosomal DNA.In this study, both sanger and 454 FLX approaches were utilised to identifyEBNA-1 sequence variation potentially relevant to immune responsein MS patients.Sanger sequencing revealed no mixed positions but identified different viralclusters mainly divergent from the reference strain B95-8.Using the sensitive 454 FLX technology, several minority species could bedetected. Additionally, four samples could be sequenced using 454FLX that could not be sequenced with sanger sequencing technology.However, these samples preferentially amplified chromosomal DNAwith low EBV reads, suggesting low EBV viral load in these samples.Most quasispecies were present at less than 1%, consistent with known limiteddsDNA viral sequence variation.Analysing sequence variation in previously described EBNA-1 epitopesdemonstrated concordant results using these two methods, withlimited evidence of viral quasispecies.ConclusionsFig.1: Semi-nested, C-terminal EBNA-1 PCR. Numbers reflect the position of the primers in B95-8.Fig. 5: Comparison of HLA-DRB1*1501 class-II epitope variation between Sanger and 454 FLX sequencing(epitope position in EBNA-1: 482-496) for 14 samples.Blue boxes = mixtures in 454 FLX sequencing. Grey boxes = variation to reference sequence B95-8.n = number of samples with this epitope.Table 2: Mixed amino acid positions for 14 samples with high coverage tested on 454 FLX.Minorities at 1%, 2%, 5% and 10% cut off. Percentage of mixed positions was calculated inrelation to the 714 investigated nucleotides of EBNA-1.ResultsEBV could successfully be amplified in 72 samples without previous culture toenrich for EBV episomes. EBV Sanger sequences clustered in four maingroups. Interestingly, most of the samples did not cluster with the EBVreference strain B59-8 (Fig.3). Genotyping of position 487 of EBNA-1identified the strain 487-threonine (n=52) and 487-alanine (n=14) asmost prevalent, with alanine also present in the B95-8 reference strain(Table 1).Fig. 2: workflow sanger and 454 FLX sequencingWe would like to thank all patients and study participants, all nurses and doctors involved as wellas staff at IIID for their assistance and input.This study is funded by the McCusker Charitable FoundationFig. 4: Maximum and average reads for 24 MS samples tested on 454 FLX.samples with mutation in the same position1 2 3 4 5 6 7 8 9 10 11 12 13 14 mixed positions % mixed positions1% positions with mutation 105 30 15 11 6 4 0 2 1 4 0 3 1 0 182 25.5%2% positions with mutation 35 17 3 3 4 2 0 1 1 0 2 1 0 0 69 9.7%5% positions with mutation 21 7 0 1 1 1 1 0 0 0 0 0 0 0 32 4.5%10% positions with mutation 19 1 1 0 0 0 0 0 0 0 0 0 0 0 21 2.9%For FLX data analysis all homopolymers not present in the consensus sequence have been excluded. Minorities present at < 1% were not taken into consideration and insertion deletions were also excluded.Fig. 3: Phylogenetic tree of C-terminal EBNA-1 MS sequences. Circled is the EBV reference strain B95-8Table 1: Genotyping position 487 ofthe EBNA-1 gene in 72 samples.

Identifying Epstein-Barr virus EBNA-1 sequence variation using 454 FLX technology in Multiple Sclerosis patient samples

https://researchrepository.murdoch.edu.au/id/eprint/36794/1/epsteinbarr%20poster.pdf

Identifying Epstein-Barr virus EBNA-1 sequence variation using 454 FLX technology in Multiple Sclerosis patient samples

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