#SANGER SEQUENCING CHROMATOGRAM VIEWER MANUAL#
Commercial software, such as SeqMan (DNASTAR Lasergene Suite, DNASTAR, Madison, WI, USA), and free graphic user interface-based applications, such as Clustal X, 1 are available for alignment, but still, they require manual intervention, which consumes time and requires skilled manpower. A consensus sequence is built either using alignment programs, such as ClustalW2, 1 or manually finding an overlapping region and then aligning the consensus with the reference sequence. The reverse sequence is first reverse complemented and then copied to a new file using the same method as for the forward sequence. The generated files need to be opened in chromatogram viewer software, such as FinchTV (Geospiza, Seattle, WA, USA) then manually, the lower quality sequences are trimmed from both ends, and the remaining good-quality sequences are made into a new file. Generally, for each sequence region, we sequence it using both forward and reverse strand-specific primers. The version described in this paper is 0.28.Īutomatic Sanger sequencing platforms, such as Applied Biosystems 3130/3500/3730 (Thermo Fisher Scientific, Waltham, MA, USA), generate chromatogram files in the “*.ab1” format. ASAP is available as Python 3.x executable at. All files are created and can be used for further analysis.
#SANGER SEQUENCING CHROMATOGRAM VIEWER FULL#
In full capabilities of Automated Mutation Analysis Pipeline (ASAP), it is able to read "*.ab1" chromatogram files through command line interface, convert it to the FASTQ format, trim the low-quality regions, reverse-complement the reverse sequence, create a consensus sequence, extract the exonic regions using a reference exonic sequence, translate the sequence in all frames, and align the nucleic acid and amino acid sequences to reference nucleic acid and amino acid sequences, respectively. Hence, we devised a rapid automated command system to filter, build, and align consensus sequences and also optionally extract exonic regions, translate them in all frames, and perform an amino acid alignment starting from raw sequence data within a very short time. This work is cumbersome and takes time, especially if the gene is large with many exons. Generally, for 1 region of a sequence, we use both forward and reverse primers to sequence that area, in that way, we have 2 sequences that need to be aligned and a consensus generated before mutation detection studies. Sanger sequencing platforms, such as applied biosystems instruments, generate chromatogram files.
0 kommentar(er)
