Human Adenovirus Genome Sequencing
This is Final report for your sample.
We finished to analyze your sample and we get the results. We confirmed two mutations and
each end-point sequence is same with reference. Regarding first mutation, we can’t confirm using
the results of experiment but it is expected T(5) though BLASTX analysis. This report includes
experiment method. Please review it and if you have any questions feel free to contact us at
anytime.
[1] For further experiments to confirm T(5) sequence of 1,862 position
SolGent provides Genescan running service for genotyping. Utilizing this method, we apply to
your sample with Snapshot typing. The process is as following below,
1.
Design primer: Synthesize the primer included T(5) sequence and then add 1bp sequence.
2.
Run: Used nucleotide have four-flourscent and then run Genescan using ABI3100.
3.
Analysis: In case of T(6), the peak will be red and T(5) will be blue.
4.
Price: The fee is $100.00. But if DNA template is mixted with T (5) and T(6), it is difficult to get
good results, too.
[2] Multiple alignment
We made the web page for it using provided three-sequence. Click below link and confirm it.
http://info.solgent.com/121210_Ad36_variation/
Figure 1. multiple alignment result web page
756-27 Dae-Duk street, Yuseong-Gu, Daejeon, 305-348, South Korea
Tel : 82-42-863-5685 |
Fax : 82-42-864-569
Human Adenovirus Genome Sequencing
[3] The process of experiment
We performed the PCR & Sanger method sequencing for your sample. We referred a few
reference (attached file; reference 5-7). And in order to good results, we used various method
which SolGent provides.
PCR and initial DNA sequencing strategy
PCR primers were generated from a consensus sequence derived from Human adenovirus 36 from
USA, complete genome (Accession Number: GQ384080, GI: 261875889, Ad36-1988). A standard
PCR with 40 primer pairs was conducted to amplify the overlapping fragments encompassing the
entire genome of Ad36 2012, except its 5' and 3' termini. PCR products, about 1.2 kb, were used
initially. Cross primer matching were used for covering gaps and for complementing sequences.
PCR reaction condition is following; Solg™ 2X Multiplex PCR Pre-Mix, denatured initially at 95 ℃
for 15 min, and then amplified at 95 ℃ for 20 sec, 55 ℃ for 40 sec and 72 ℃ for 1 min for 35
cycles, followed by a final extension at 72 ℃ for 3 min and stored at 4 ℃.
Potential primers for PCR amplifications and sequencing were
synthesized by
Bioneer
(http://www.bioneer.com/).
*attached file; primer list.xls (design condition: Length: 21~22, GC(%): 35.0~60.0, Tm(C): 40~65.0).
Figure 2. Reference mapping of sequencing read
756-27 Dae-Duk street, Yuseong-Gu, Daejeon, 305-348, South Korea
Tel : 82-42-863-5685 |
Fax : 82-42-864-569
Human Adenovirus Genome Sequencing
End sequencing
A method similar to that which uses a 5'/ 3' rapid amplification of cDNA ends kit was followed.
1) 3' end sequencing
First,
single
strand
DNA
synthesized
with
phosphorylated
PCR
primer
(5'-
PAGCACAGCAGTACAAGCGC-3') and self-ligated using T4 DNA ligase (Promega). Subsequently,
inverse PCR were performed using ligated single strand DNA as template with internal primers (5'GTTCCCACGTTACGTCACTT-3', 5'-CGAGGAAGTGACAACGCGGAA-3').
Figure 3. 3’-end analysis scheme
2) 5' end sequencing
756-27 Dae-Duk street, Yuseong-Gu, Daejeon, 305-348, South Korea
Tel : 82-42-863-5685 |
Fax : 82-42-864-569
Human Adenovirus Genome Sequencing
First, PvuII restriction enzyme (Jena Bioscience, Germany) digest DNA template. Then in the
presence of T4 DNA ligase (Promega), the blunt-ended DNA templates were ligated. Subsequently,
inverse PCR were performed with internal primers (5'-CACTTGCATCCGCCCAGAAT-3' and 5'CGAAAACTGAATGAGGAAGTG-3') for amplification of the 5'-terminal DNA sequences to obtain the
sequencing templates.
Figure 4. 5’-end analysis scheme
DNA sequencing and analyses
The amplified fragments were purified by 1 % agarose gel electrophoresis by using a SolGent™
756-27 Dae-Duk street, Yuseong-Gu, Daejeon, 305-348, South Korea
Tel : 82-42-863-5685 |
Fax : 82-42-864-569
Human Adenovirus Genome Sequencing
PCR Purification Kit (Solgent, Daejeon, Korea). The sequencing reactions were performed
bidirectionally with the appropriate primers and cycle sequencing kits (ABI Prism BigDye
Terminator, version 3.1) and were then resolved with a model 3730XL DNA Analyzer (Applied
Biosystems, Foster City, CA, USA).
We used special analysis method with difficult sequence caused by repeated poly(N).
The purified products were cloned into T-Blunt vector (T-Blunt™ PCR Cloning Kit, Solgent) and
reacted rolling circle amplification (RCA, SEQ-TempliGen™ RCA Kit, Solgent). Phred-Phrap-Consed
program [1]-[3] were used for sequence assembly and editing assembled sequences.
[1] Ewing B, Green P. 1998. Base-calling of automated sequencer traces using phred. II. Error
probabilities. Genome Res. 8: 186– 194.
[2] Ewing B, Hillier L, Wendl MC, Green P. 1998. Base-calling of automated sequencer traces using
phred. I. Accuracy assessment. Genome Res.8: 175– 185.
[3] Gordon D, Abajian C, Green P. 1998. Consed: a graphical tool for sequence finishing. Genome
Res. 8: 195– 202.
[4] Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. 2009.
BLAST+: architecture and applications. BMC Bioinformatics 10:421.
[5] Seto, J.; Walsh, M.P.; Mahadevan, P.; Zhang, Q.; Seto, D. 2010. Applying Genomic and
Bioinformatic Resources to Human Adenovirus Genomes for Use in Vaccine Development and for
Applications in Vector Development for Gene Delivery. Viruses 2, 1-26.
[6] Yang Z, Zhu Z, Tang L, Wang L, Tan X, Yu P, Zhang Y, Tian X, Wang J, Zhang Y, et al. 2009.
Genomic analyses of recombinant adenovirus type 11a in China. J Clin Microbiol 47(10):3082-90.
[7] Lauer KP, Llorente I, Blair E, Seto J, Krasnov V, Purkayastha A, Ditty SE, Hadfield TL, Buck C,
Tibbetts C, Seto D. 2004. Natural variation among human adenoviruses: genome sequence and
annotation of human adenovirus serotype 1. J Gen Virol 85(Pt 9):2615-25.
756-27 Dae-Duk street, Yuseong-Gu, Daejeon, 305-348, South Korea
Tel : 82-42-863-5685 |
Fax : 82-42-864-569