Sequencing reactions were analyzed on an ABI 3730XL automated DNA sequencer and sequence trace files were exported into Sequencher 4.7 (GeneCodes, Ann Arbor, MI).
Raw sequence trace data from the 3100 sequencer were processed in an automated pipeline, the trace2dbEST package [ 72].
Sequence trace curves were collected on the computer controlling the sequencer.
Brinkman, E. K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition.
Brinkman, E.K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition.
Sequence trace files were aligned and quality checked using SeqMan package in DNASTAR Lasergene 6 software suite.
Frame shifts and point mutations were corrected or designated 'authentic' based on manual examination of the sequence trace files.
DNA sequence trace files were aligned with the program Sequencher™ (Gene Codes Corp ,MI), and analyzed for levels of sequence divergence as described below.
Tumor samples which were repeat positive for an aberrant melting curve were sequenced in duplicate, and the resulting sequence trace files were analyzed for mutations using the phrap/phred/consed software package (www.phrap.org).
Raw ABI sequence trace files from selected clones in this study as well as all trace files from the JGI were assembled into contigs using ContigExpress, with criteria of 80% identity and 40 bp overlap.
Clicking on individual SNP provides access to the sequence trace files, sequence alignments, the primers used for analysis, the genomic position and minor allele frequency data for each SNP.
