Abi 3730 dna sequencer

Three (range 0–4) viral isolates identified as influenza B per month during 2011–2017 were randomly selected for sequencing. Viral nucleic acids were extracted from cell culture and the complete fragment of hemagglutinin (HA) gene and neuraminidase (NA) gene were sequenced. Specific primers used for target HA and NA gene amplification by RT-PCR were designed (Table S4), referencing to the HA and NA gene of strain B/Yamagata/16/88. The RT-PCR was performed using the One Step RT-PCR Kit (Qiagen, Germany) according to the handbook. The PCR products were analyzed using QIAxcel capillary electrophoresis (Qiagen, Germany) and sequenced by ABI 3730 DNA sequencer (PE-Applied Biosystems, Foster City, CA).
Multiple sequence alignment was performed using Clustal W (version 1.83). Genome sequences included in this study were submitted in the Global Initiative on Sharing Avian Influenza Data (GISAID)/GISAID accession numbers: EPI1153147-EPI1153378 (Table S4). A total of 83 HA and 83 NA sequences derived ILI outpatients from 2011 to 2017 were analyzed and 60 strains were downloaded from NCBI and GISAID using as reference strains in this study. The phylogenetic trees were constructed using MEGA software (version 6.06) applying the neighbor-joining method with 1000 bootstrap replicates.

Methylation Primer Express Software V1.0 was used to design bisulfite sequencing PCR (BSP) primers, which are provided in Table S1. DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen, CA, USA) according to the manufacturer’s protocol. We acquired bisulfite converted DNA using the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s protocols. PCR was carried out with the ZymoTaq PreMix (Zymo Research, CA, USA) according to the manufacturer’s specifications. The PCR products were subsequently purified using the DNA Clean & Concentrator - 25 Kit (Zymo Research). Subsequently, the PCR products were cloned into a TA vector (Invitrogen, Carlsbad, CA, USA). Ten effective subclones were selected for each gene, and successful cloning was subsequently confirmed by analyzing the sequence data (BiQ Analyzer V2.0) obtained with an ABI 3730 DNA sequencer (Applied Biosystems, Foster City, CA)29 (link).

To validate the results of HRM analysis, the HRM-PCR products were purified using Multifunction DNA Purification Kit (Bioteke, China). And then the purified PCR products were sequenced in both directions with the same PCR primers on an ABI 3730 DNA Sequencer (Applied Biosystems). The CodonCode Aligner 4.0.4 (CodonCode Co., USA) was used to proofread, assemble the contigs, and generate consensus sequences. Complete ITS2 sequences were retrieved according to hidden Markov model based model analysis. The sequence alignment was performed with Clustal W. The tree-based method was used for species identification analyses: the neighbor-joining (NJ) tree was conducted by MEGA 5.0 with 1000 bootstrap replicates, and the bootstrap value above 50% was shown. All the sequences were also submitted to DNA barcoding system for identifying herbal medicine (http://www.tcmbarcode.cn/en/) to verify the HRM results.

To confirm identity of RABV in central nervous system (CNS) tissue of euthanized animals with the initial inoculum and to identify any potential selection of escape mutations, total RNA was extracted from the CNS tissue samples using TRIZol reagent (Ambion, Carlsbad, CA, USA) according to the manufacturer’s recommendations. The RT-PCR was performed as described elsewhere [16 ]. The RT-PCR products were purified and subjected to direct sequencing on an ABI 3730 DNA Sequencer (Applied Biosystems, Carlsbad, CA, USA). The complete and partial nucleotide G gene sequences were assembled and converted into amino acid sequences using the Bio Edit program, v.7 (Ibis Biosciences, Carlsbad, CA, USA) [17 ]. Amino acid sequences of the aligned MAb binding epitopes were compared across the dataset.

Based on the SNP and linkage disequilibrium analysis of TNNT2 in our recent study in DCM patients [12 ], an approximately 4-kilo-base (kb) fragment of TNNT2 located at chr.201333582-201337484 was selected, and the fragment included exons 6, 7, 8, 9, and 10 and introns 5, 6, 7, 8, 9, and 10. The TNNT2 fragment was amplified by PCR with primers (primer sequences available on request). Products were then sequenced using an ABI 3730 DNA Sequencer (Applied Biosystems, Foster City, CA). The DNA sequence was viewed and analyzed using the Sequencher computer program (Gene Codes Corporation, Ann Arbor, MI).