1. Rapid Decoding of Sequence-Specific Nuclease-Induced Heterozygous and Biallelic Mutations by Direct Sequencing of PCR Products 
Xingliang Ma, Letian Chen, Qinlong Zhu, Yuanling Chen, Yao-Guang Liu 
Molecular Plant 
Volume 8, Issue 8, Pages (August 2015)
DOI: /j.molp
Copyright © 2015 The Author Terms and Conditions

2. Figure 1
Types of Non-homozygous Mutations in Plants Induced by CRISPR/Cas9 and the Procedure for Decoding Mutated Sequences from Direct Sequencing of a PCR Product.
(A) Representative types of heterozygous (types 1-3), biallelic (types 4 and 5), and triallelic/chimeric (type 6) mutations in rice (types 1–5) and Arabidopsis (type 6) induced by our CRISPR/Cas9 system, which produce superimposed sequencing chromatograms. Biallelic mutations with single-base insertion are not shown. Lowercase red letters indicate nucleotide insertion (In), and a red dash shows base deletion (Del). Arrows show where overlapping peaks of sequencing chromatograms are produced and degenerate sequences are deduced.
(B) Nucleotides were deduced according to the peak colors in the sequencing chromatogram and overlapping nucleotides were converted into degenerate bases using the standard degenerate nucleotide symbols. The anchor site (underlined) borders the first degenerate base. The degenerate sequence (query) was searched against the wild-type reference sequence with a sequence analysis program to find the matched sequence (boxed). The anchor site (underline) is identified manually or by the same program. By linking the query-matched sequence to the anchor site, the first allele (allele 1) was decoded. Then, sequence of allele 2 was obtained by subtracting the allele 1 nucleotides (blue) from the degenerate bases. Alignment of the decoded sequences (type 4 mutation) is shown in (A).
Molecular Plant 2015 8, DOI: ( /j.molp )
Copyright © 2015 The Author Terms and Conditions