Open AccessComparative next-generation sequencing of adeno-associated virus inverted terminal repeats
Author(s) -
Karl Petri,
Raffaele Fronza,
Gabriel Rinaldi,
Christine Käppel,
Ali Nowrouzi,
R. J. Linden,
Els Henckaerts,
Manfred Schmidt
Publication year - 2014
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/000114170
Subject(s) - amplicon , biology , dna sequencing , adeno associated virus , palindrome , sequence (biology) , genetics , inverted repeat , computational biology , microbiology and biotechnology , polymerase chain reaction , dna , gene , recombinant dna , genome , vector (molecular biology)
The inverted terminal repeats (ITRs) of adeno-associated virus (AAV) are notoriously difficult to sequence owing to their high GC-content (70%) and palindromic sequences that result in the formation of a very stable, 125 bp long, T-shaped hairpin structure. Here we evaluate the performance of two widely used next-generation sequencing platforms, 454 GS FLX (Roche) and MiSeq Benchtop Sequencer (Illumina), in analyzing ITRs in comparatively sequencing linear amplification-meditated PCR (LAM-PCR) amplicons derived from AAV-concatemeric structures. While our data indicate that both platforms can sequence complete ITRs, efficiencies (MiSeq: 0.11% of sequence reads; 454: 0.02% of reads), frequencies (MiSeq: 171 full ITRs, 454: 3 full ITRs), and rates of deviation from the derived ITR consensus sequence (MiSeq: 0.8%–1.3%; 454: 0.5%) did differ. These results suggest that next-generation sequencing platforms can be used to specifically detect ITR mutations and sequence complete ITRs.