Species identification in mixed tuna samples with next-generation sequencing targeting two short cytochrome b gene fragments.

Conventional Sanger sequencing of PCR products is the gold standard for species authentication of seafood products. However, this method is inappropriate for the analysis of products that might contain mixtures of species, such as tinned tuna. The purpose of this study was to test whether next-generation sequencing (NGS) can be a solution for the authentication of mixed products. Nine tuna samples containing mixtures of up to four species were prepared and subjected to an NGS approach targeting two short cytochrome b gene (cytb) fragments on the Illumina MiSeq platform. Sequence recovery was precise and admixtures of as low as 1% could be identified, depending on the species composition of the mixtures. Duplicate samples as well as two individual NGS runs produced very similar results. A first test of three commercial tinned tuna samples indicated the presence of different species in the same tin, although this is forbidden by EU law.

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

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.

The effect of Cu(II)-loaded brushite scaffolds on growth and activity of osteoblastic cells.

Bone substitute materials such as calcium phosphate cements (CPC) are frequently used as growth factor carriers for the stimulation of osteoblast-formation around an implant. However, biological modification based on delicate protein factors like extracellular matrix proteins or growth factors is subject to a number of shortcomings like the need for storage below room temperature and cost of production. The aim of this study was to investigate ionic modification as an alternative bioinorganic route for implant modification. Although it is known that Cu(II) plays a role in angiogenesis and bone formation, not all involved processes are well understood yet. In this study the in vitro effect of Cu(II) on growth and activity of osteoblastic cells seeded on brushite (CaHPO(4) · 2 H(2) O) scaffolds as well as on glass discs was investigated. The results show that Cu(II) enhances cell activity and proliferation of osteoblastic cells on CPC and furthermore affects the expression of several bone specific proteins such as bone sialo protein or osteocalcin. Therefore, the modification of CPC with Cu(II) may offer a promising alternative to protein based modification to stimulate cellular activity for an improved bone healing.