Seqman program

The polymerase chain reaction (PCR) was performed from DNA using primers that cover the entire coding region from β-myosin heavy chain (MYH7), myosin binding protein C (MYBPC3), and troponin T (TNNT2) genes (primer sequences available upon request). After PCR, the fragments were purified with the ExoSAP-IT® enzyme (GE Healthcare). The sequencing reaction was performed with BigDye Terminator V3.1 Cycle sequencing Kit® (Life Technologies) and EDTA/ethanol precipitation protocol. The samples were sequenced in an automatic sequencer ABI3500xl (Life Technologies).
The sequences were evaluated with the SeqMan program (DNASTAR Lasergene, Madison, WI) and compared with the reference sequence in the NCBI database. For MYH7, MYBPC3, and TNNT2, the references were, respectively, NM_000257.2, NM_000256.3, and NM_000364.2 [8 ]. When an undescribed mutation was found, we used bioinformatics algorithms to evaluate the pathogenic potential of the alteration. The SIFT [9 (link)] and Polyphen[10 (link)] programs were used only for substitutions, and the MutationTaster[11 (link)] was used to analyze deletions, insertions, and intron alterations. A mutation was labeled as pathogenic if (1) it had been previously described as causing disease; (2) it generated an aminoacid change and was considered pathogenic by all 3 programs above or in 2 programs but the aminoacid was conserved.

Viral RNA was extracted from allantoic fluid using TRIzol Reagent (Invitrogen Carlsbad, CA, USA) and reverse transcribed into cDNA using the primer Uni12 (5′-AGC RAA AGC AGG-3′). PCR products of H5N6 virus HA and NA fragments were amplified using specific viral primers as described by Hoffmann et al.^{24 (link)}. The PCR products were purified and sequenced by Comate Bioscience Company Limited, and sequence data were analyzed with the SEQMAN program (DNASTAR, Madison, WI, USA). All reference sequences used in this study were obtained from the National Center for Biotechnology Information (NCBI) GenBank database. Phylogenic analysis was performed by the distance-based neighbor joining method using MEGA7.0.21 software (DNAStar, Inc.).

According to the published genomic sequences of CAV reference strains in the GenBank, four pairs of primers were designed and synthesized to amplify the full length of CAV genome (Table 1). The primers were synthesized by Sangon Biotech Co.,Ltd (Shanghai, China), and high fidelity DNA polymerase was used for amplification. The total DNA was extracted from the cultured cell supernatants of 33 CAV positive samples. PCR conditions were the same as described previously. Then, amplified products were sequenced and sequence assembly was carried out using the SeqMan program included in the DNASTAR software package (DNAStar Inc., Madison, WI, USA).

A QIAGEN Viral RNA Isolation Kit was used to extract vRNA from 140 μL allantoic fluid from infected SPF embryonated eggs. The isolated RNA was eluted into 30 μL diethylpyrocarbonate-treated water. Two-step RT-PCR was employed to amplify each viral gene segment. The first-strand cDNA was transcripted by using reverse transcriptase M-MLV (TaKaRa, Dalian, China) with universal primer (5′-AGCAAAAGCAGG-3′) for influenza A virus in a final volume of 20 μL per manufacturer’s protocol. Eight fragments (PB2, PB1, PA, HA, NP, NA, M and NS) of each virus were amplified respectively by using the Hoffman universal primers for influenza A virus as described previously31 (link). Amplicons of the appropriate sizes were subsequently gel purified by using a DNA Gel Extraction Kit (Axygen, Hangzhou, China). The purified PCR products were sequenced by the GENEWIZ Biotechnology Company using the Sanger sequencing methodology. Sequences were assembled by using SEQMAN program (DNASTAR, Madison, WI, USA).

Sequence assembly and manual editing were performed using the SeqMan program (DNASTAR, Madison, WI, USA). The nucleotide (nt) sequence identities were calculated by the MegAlign program available within the Lasergene software package (version 7.1, DNAstar) [22 (link)]. All the sequences obtained in this study have been submitted to GenBank under the accession numbers OK617223–OK617250 and OK632480–OK632507.
The best-fit evolutionary model of nt substitution was determined using jModelTest [23 (link)]. The maximum-likelihood (ML) trees were constructed based on the general time-reversible (GTR) nucleotide substitution model and the optimized parameters of gamma (Γ)-distribution and proportion of invariable sites (i.e., GTR + Γ + I) with bootstrap support values calculated from 100 replicates implemented in MEGA X [24 (link)]. All phylogenetic trees were mid-point rooted for purposes of clarity only.