RnaseIII and T4 Polynucleotide Kinase Sequence Biases and Solutions During RNA-Seq Library Construction

Background: RNA-seq is a next generation sequencing method with a wide range of applications including single nucleotide polymorphism (SNP) detection, splice junction identification, and gene expression level measurement. However, the RNA-seq sequence data can be biased during library constructions resulting in incorrect data for SNP, splice junction, and gene expression studies. Here, we developed new library preparation methods to limit such biases. Results: A whole transcriptome library prepared for the SOLiD system displayed numerous read duplications (pile-ups) and gaps in known exons. The pile-ups and gaps of the whole transcriptome library caused a loss of SNP and splice junction information and reduced the quality of gene expression results. Further, we found clear sequence biases for both 5' and 3' end reads in the whole transcriptome library. To remove this bias, RNaseIII fragmentation was replaced with heat fragmentation. For adaptor ligation, T4 Polynucleotide Kinase (T4PNK) was used following heat fragmentation. However, its kinase and phosphatase activities introduced additional sequence biases. To minimize them, we used OptiKinase before T4PNK. Our study further revealed the specific target sequences of RNaseIII and T4PNK. Conclusions: Our results suggest that the heat fragmentation removed the RNaseIII sequence bias and significantly reduced the pile-ups and gaps. OptiKinase minimized the T4PNK sequence biases and removed most of the remaining pile-ups and gaps, thus maximizing the quality of RNA-seq data.National Institute on Alcohol Abuse and Alcoholism (NIAAA) AA12404, AA019382, AA020926, AA016648National Institutes of Health (NIH) R01 GM088344Waggoner Center for Alcohol and Addiction Researc

Accuracy of structure-based sequence alignment of automatic methods

<p>Abstract</p> <p>Background</p> <p>Accurate sequence alignments are essential for homology searches and for building three-dimensional structural models of proteins. Since structure is better conserved than sequence, structure alignments have been used to guide sequence alignments and are commonly used as the gold standard for sequence alignment evaluation. Nonetheless, as far as we know, there is no report of a systematic evaluation of pairwise structure alignment programs in terms of the sequence alignment accuracy.</p> <p>Results</p> <p>In this study, we evaluate CE, DaliLite, FAST, LOCK2, MATRAS, SHEBA and VAST in terms of the accuracy of the sequence alignments they produce, using sequence alignments from NCBI's human-curated Conserved Domain Database (CDD) as the standard of truth. We find that 4 to 9% of the residues on average are either not aligned or aligned with more than 8 residues of shift error and that an additional 6 to 14% of residues on average are misaligned by 1–8 residues, depending on the program and the data set used. The fraction of correctly aligned residues generally decreases as the sequence similarity decreases or as the RMSD between the C_{<it>α </it>}positions of the two structures increases. It varies significantly across CDD superfamilies whether shift error is allowed or not. Also, alignments with different shift errors occur between proteins within the same CDD superfamily, leading to inconsistent alignments between superfamily members. In general, residue pairs that are more than 3.0 Å apart in the reference alignment are heavily (>= 25% on average) misaligned in the test alignments. In addition, each method shows a different pattern of relative weaknesses for different SCOP classes. CE gives relatively poor results for <it>β</it>-sheet-containing structures (all-<it>β</it>, <it>α</it>/<it>β</it>, and <it>α</it>+<it>β </it>classes), DaliLite for "others" class where all but the major four classes are combined, and LOCK2 and VAST for all-<it>β </it>and "others" classes.</p> <p>Conclusion</p> <p>When the sequence similarity is low, structure-based methods produce better sequence alignments than by using sequence similarities alone. However, current structure-based methods still mis-align 11–19% of the conserved core residues when compared to the human-curated CDD alignments. The alignment quality of each program depends on the protein structural type and similarity, with DaliLite showing the most agreement with CDD on average.</p

インターネット観光情報の構成と選好度調査

本研究では佐世保市に居住する観光需要者を中心にインターネット観光情報の重要項目と満足度を調査した。研究の結果、インターネット観光情報の利用形態やその他の観光関連情報は費用に関する情報が最も重要とされていることが分かった。インターネット観光情報の提供においては観光商品の品質や個人情報の保護は不満足であることで分析され、何より観光需要者を理解し、彼らのニーズを最大限に受容できるような観光情報の提供が必要であることが分かった。また、観光者の期待と満足の不一致を克服するために、観光者の関心を高めることができる魅力的な観光商品の開発と新しい観光情報を持続的に提供することも重要である

Direct Detection of the Biological Toxin in Acidic Environment by Electrochemical Impedimetric Immunosensor

This study describes the direct detection of the biological toxin (Ricin) in acidic environment without pH adjustment by hydrophobically modified electrochemical impedance immunosensor (EII). The nano-porous aluminum substrate for EII was hydrophobically modified via self-assembled monolayer (SAM) of APTES. Biosensor for the detection of the Ricin was fabricated by the covalent cross-linking of antibody (Ab) with APTES-SAM. The immunoreactions between the immobilized Ab and the biological toxin in several diagnostic solutions were monitored by the electrochemical impedance spectroscopy (EIS) under the polarization of EII versus reference electrode. EII could detect the presence of the biological toxin in acidic foods in 20 mins without pH adjustment. The negatively charged ions including hydroxides would be adsorbed on the hydrophobic body of APTES-SAMs by the polarization during EIS analysis, and offset the effect of acids on the immunological activity of the immobilized Ab. It suggested that the adsorption of negatively charged ions helped to keep the immunological activities of the immobilized Ab on EII in acidic environment. Proposed mechanism of the localized pH adjustment that makes possible immunoreaction occurrence in low pH sample matrix is briefly discussed