Chapter 2: Methods
2.5. HDV sequence identification
elsewhere in details [141]. A high resolution assay was done on some patients to define a four-digit HLA type or sub-groups.
2.5. HDV sequence identification
2.5.1. L-HD Ag open reading frame amplification
Reverse Transcriptase: Ten μl of extracted RNA was used to make the complementary DNA (cDNA). First the RNA was denatured in the presence of 1 μl reverse primer (771R, Figure 1B) at 98°C for 5 min and then at 62°C for another 5 min, and then the cDNA was synthesized by adding 4 μl 5X reaction buffer, 4 μl dNTPs (2.5mM), 1 μl MMLV-RT polymerase (Promega, USA) and 0.5 μl of RNAse inhibitor in a total volume of 20 μl at 37°C for 1 hour.
First PCR: A reaction mix of 45 μl containing 23.75 μl ddH2O, 10 μl 5X reaction buffer, 5 μl primer 891(10pmol), 5 μl primer 339R(10pmol), 1 μl dNTPs (10mM) and 0.25 μl GoTaq (Promega, USA) was made and added to 10 μl of cDNA obtained from RT-PCR. Then the reaction was done under the following thermal profile: denaturation at 94°C for 10 Min. and a 35 cycle of 94°C for 30 Sec, 54°C for 45 Sec., and 72°C for 90 Sec. and then an elongation step at 72°C for 7 Min. and finally stop at 4°C.
Nested PCR: The same reaction mixture as for the 1st PCR was prepared but with primers 912 and 1674R and then 5 μl of the 1st PCR product was added to each tube to undergo a nested PCR applying the following thermal conditions: denaturation at 94°C for 10 Min. and a 35 cycle of 94°C for 30 Sec, 56°C for 45 Sec., and 72°C for 90 Sec. and then an elongation step at 72°C for 7 Min. and finally stop at 4°C.
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Figure 2.1(A) Schematic representation of HDV genome and the location of PCR amplified fragments. (B) List of primers used for reverse transcription (RT), amplification of L-HD Ag open reading frame and sequencing.
2.5.2. PCR product purification and preparation of positive samples for sequencing
The final positive PCR products were purified using either QIAquickSpin kit or QIAquick Gel Extraction Kit (see Appendix section for the detailed instruction). To avoid interfering the sequencing by unspecific PCR products which were shorter or longer than the region of interest (L-HD Ag coding region), Gel extraction protocol were performed. Briefly, the total volume of the PCR product of each sample was loaded on a 1% agarose gel and run for an hour and then the correct band of each sample was cut out. The PCR product of this piece of gel was extracted and purified exactly according to the instruction of QIAquick Gel Extraction Kit (see Appendix II). The concentration of the purified samples was measured by NanoDrop® and finally they were prepared and sequenced at LGC Company on the ABI 3730 XL platforms for traditional Sanger sequencing.
2.5.3. Phylogenetic analysis and genotyping of HDV
From each patient/time-point both forward and reverse sequences were evaluated using Geneious program version 7.0.6 (Biomatters, Auckland, New Zealand) and exported as FastA file. A master FastA file of full-length L-HD Ag sequences from this study and 53 reference sequences (HDV genotypes 1-8) from NCBI GenBank were used to perform multi sequence alignment using ClustalX2 method [142] integrated to the Geneious program. A Maximum Likelihood (ML) phylogenetic tree was constructed under the Tamura-Nei substitution model using MEGA software v6 [143]. To address the reliability of pairwise
27 comparison and phylogenetic tree analysis, bootstrap test was performed with 1,000 replicates. All sites containing alignment gaps and missing information had been removed before the calculation started by selecting “Complete-deletion” option under “Gaps/Missing Data Treatment”. In order to improve the likelihood of the phylogenetic tree, “Nearest-Neighbor-Interchange” was applied as a ML heuristic method. Genotypes of strains were determined by comparison to the full-length of HD Ag reference sequences, representing all HDV genotypes.
2.5.4. Longitudinal study
On closer examination of sequencing data, we observed a few number of sites showing single nucleotide polymorphisms (SNPs) (Figure 2.2). Therefore, we were interested to know whether these changes are correlated with immune pressure at these sites promoting evolution of the virus in those HDV infected patients over time. To investigate this, we analyzed longitudinal samples from 24 patients with chronic HDV infection for varying time lengths, from 3 months up to 6 years. In this study also the only ORF of HDV encoding for L-HD Ag was amplified and sequenced. Then the post-infection mutation rate was determined over time. Evolutionary distance was also calculated within the individual patients and within the whole group of the patients.
Figure 2.2 Schematic illustration of single nucleotide polymorphisms (SNPs) within the L-HD Ag coding region.
2.5.5. HDV genome database
To facilitate molecular analysis of this study, a local database was developed using Geneious program version 7.0.6 (Biomatters, Auckland, New Zealand). This database consists of all L-HD Ag sequences from this study and all thus far experimentally identified and submitted HDV isolates retrieved from GenBank available at https://www.ncbi.nlm.nih.gov/genbank. This database also includes of all thus far studied partial L-HD Ag sequences as well as HDV full genome sequences.
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2.5.6. Deep sequencing by pyrosequencing
In order to identify variations at lower detection level of direct sequencing technology, we applied next generation sequencing by ultra-deep pyrosequencing (UDPS), to pin down quasispecies in specific region.
All nucleotide (nt) numbering positions are according to HDV sequence ID: dbj|D01075.1 | HPDCGDA. One µl of the first PCR product obtained by primers 891fw and 339rv was processed by a second run of PCR by primers flanking positions 956 and 1360 which included M13 fagus universal tails in 5’ ends followed by specific HDV sequences:
M13HDV956forward: downstream) followed by a Roche's Validated Multiplex IDentifier (MID, indicated in italics), and with oligoA or B at 5' or 3' end of the upstream or downstream primer respectively (in
OligoAMIDM13fw and OligoBMIDM13rv primers were preloaded and lyophilized in 96 wells ready to use PCR plates. Therefore, the use of M13 universal primers allow the incorporation of MID sequences by means of these PCR plates preloaded with OligoAMIDM13fw and OligoBMIDM13rv primers, avoiding sample identification errors.
The final 515 bp amplicon included a 405 nt HDV specific sequence in which, once discarding primer sequences, 362 nt HDV sequences were analyzed (positions 977 to 1338) by UDPS. This sequence covered HD Ag amino acids 89 to 208. Further general details on this method are discarded elsewhere [144]. Since there was no NGS facility available in our institute, this part of experiments was designed and performed together with our collaborator at Vall d'Hebron Hospital, in Barcelona, Spain.