linear-amplification-mediated-pcr-localization-genetic-elements

Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with ...

National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ)

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.

Comparative next-generation sequencing of adeno-associated virus inverted terminal repeats.

Reply to: NGS library preparation may generate artifactual integration sites of AAV vectors.

Introduction: This diffusion tensor MR imaging (DTI) study aimed to clarify the relationship of peripheral nerves and soft tissue tumors (STT) in 3D to optimize subsequent treatment. Methods: 26 consecutive STT patients (histologically malignant n=10, intermediate n=3, and benign n=13) underwent 3 Tesla MRI using an echoplanar DTI sequence. Deterministic tractography was performed. Fractional Anisotropy (FA) values were measured within peritumoral and distant ROIs. Results: Tractography depicted the 3D course of the sciatic (n=12), femoral (n=2), tibial (n=7), fibular (n=2), median (n=1), musculocutaneous (n=1), and ulnar (n=1) nerves in a regular (n=8/18, 44.4%) or thinned (n=7/18, 38.9%) fashion. The lowest peritumoral FA values, abrupt thinning, and/or complete discontinuity of trajectories were found in 2 cases with histologically proven tumoral nerve infiltration. Discussion: DTI clarifies the 3D topography between major peripheral nerves and STT and may be helpful in the assessment of peripheral nerve infiltration by malignant tumors. © 2014 Wiley Periodicals, Inc.

Article	Year
Comparative next-generation sequencing of adeno-associated virus inverted terminal repeats. BioTechniques\| PubMed ID: 24806228	2014
Reply to: NGS library preparation may generate artifactual integration sites of AAV vectors. Nature medicine\| PubMed ID: 24901561	2014
Peripheral Nerve Tractography in Soft Tissue Tumors: A preliminary 3 Tesla DTI study. Muscle & nerve\| PubMed ID: 24916781	2014
Vector integration and tumorigenesis. Human gene therapy\| PubMed ID: 24950086	2014

Manfred Schmidt

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