DNA Sequencing Protocol Notes

ABI 3130xl genetic analyzer information

The facility DNA sequencing system was upgraded in January 2008 from the ABI 3100 to the ABI 3130xl. Following is some information on the system:

Protocols

Recommended DNA purification kits

Notes on analysis of results with sequence analysis software V5.3

  1. KB basecaller V1.1:
    1. A novel basecaller algorithm that performs basecalling for pure and mixed base calls.
    2. Generation of quality values (QV) to provide basecall accuracy information for pure and mixed base calls.
      • QV = -10 log10 (Pe)Where Pe is the probability of error
      • The KB basecaller generates QVs from 1 to 99, and the QVs are optionally displayed as bars above each base in the Sequences and Electropherograms views.
        QVBar colorSignificance
        0 to 14RedNot acceptable
        15 to 19YellowNeed manual review
        20BlueAcceptable
  2. Clear range is the region of a DNA sequence that remains after excluding the low quality or error-prone sequence at both 5’and 3’ ends. The data excluded from the clear range is shown in gray.
  3. Multiple formats of output sequence files:
    • Analyzed ABI files (.ab1)
    • Text file in ABI or Fasta (.seq)
    • Phred (.phd.1) files
    • Standard chromatogram format (.scf) files
    • Acrobat (.pdf)

    Generally we provide ABI and Fasta files, but if clients require other type of the sequence files, we can re-analyze data, and then send phred or chromatogram files.

Software for sequence file analysis

  • Finch TV to view, edit, print, and blast your sequence data.
  • ApE-A Plasmid Editor, to view, edit, and analyze data, including alignment of multiple sequences.
  • Sequence Scanner from ABI to edit and view both analyzed and raw sequence data, clear range at 5’ and 3’ ends, QVs and QV bars for the sequences, and export traces in multiple formats including pdf, phd.1, seq etc.

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