Journal
BIOTECHNIQUES
Volume 56, Issue 5, Pages 269-+Publisher
FUTURE SCI LTD
DOI: 10.2144/000114170
Keywords
adeno-associated virus; inverted terminal repeat; next-generation sequencing
Funding
- German Cancer Aid [110410]
- European Commission [FP7-HEALTH-2010-261506]
- MRC [G1001764] Funding Source: UKRI
- Medical Research Council [G1001764] Funding Source: researchfish
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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 FOR (LAMPCR) 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.
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