大致分为以下步骤：
1 PGA 批量注释，并得到两个IR区位置
2 导入geneious内，与ref-seq align，标出LSC与SSC位置，注释区间需根据IR区的位置修改。
3 使用geneious内genbank submit上传，选择save到本地，复制并保存错误记录。查看其中错误。或使用如下链接，保存Validation查看修改。https://chlorobox.mpimp-golm.mpg.de/GenBank2Sequin.html
错误修改办法
| 错误代码 | geneious 插件官方说明 | 修改方式 |
|---|---|---|
| ERROR: valid [SEQ_FEAT.NoStop] Missing stop codon FEATURE: CDS: ribosomal protein S12 | Missing stop codon：This error message occurs when the automatic translation of a CDS annotation does not end with a stop codon and the CDS is not labelled as partial. Check that the frame and translation table for this annotation are correct, and, if not, change these properties. Alternatively, if this CDS is a partial CDS sequence, open the Edit Annotations window and double click on the annotation interval. In the window that pops up, specify that the 3′ end of the annotation is truncated. Some mitochondrial genomes contain CDS’s that do not include a DNA-encoded stop codon. A stop codon is created after transcription by polyadenylation, where the terminal ‘T’ or ‘TA’ of the transcript is combined with the poly-A tail to form a ‘TAA’ stop codon. These non-standard CDS’s require an extra property attached to the CDS to be acceptable for submission. You need to add the property transl_except to the CDS. The figure below shows the format for this property, pos:XXXX indicates the position of the terminal ‘T’ nucleotide of the CDS, aa:term indicates that two A’s should be appended to the 3′ terminus. If the CDS ends TA, then the Value: would be (pos:1..2, aa:term) where 1 is the sequence position of the ‘T’ nucleotide and 2 is the position of the ‘A’. | 拉长注释区，使出现TAG，TAA，TGA等终止密码子。 |
| ERROR: valid [SEQ_FEAT.NoProtein] No protein Bioseq given FEATURE: CDS: | Missing qualifiers or features：Annotation qualifiers should be written in lowercase letters, otherwise they will not be recognised. For example, if you write the “product” qualifier, which is required for CDS and other amino acid feature (e.g. tRNA) annotations, with a capital “P” (Product) you will get error messages such as:Missing encoded amino acid qualifier in tRNA feature,or No protein BioSeq given FEATURE:CDS and Expected CDS Product absent | 在CDS的product中写入编码蛋白功能 |
| ERROR: valid [SEQ_FEAT.MissingCDSproduct] Expected CDS product absent FEATURE: CDS: | 同上 | 在CDS中添加一行codon_start，值写入为1 |
| ERROR: valid [SEQ_FEAT.MissingCDSproduct] Expected CDS product absent FEATURE: CDS: | 同上 | 在CDS的product中写入编码蛋白功能 |
| ERROR: valid [SEQ_FEAT.StartCodon] Illegal start codon used. Wrong genetic code [1] or protein should be partial FEATURE: CDS: ATP synthase CF0 subunit IV | Illegal start codon used：NCBI will automatically generate the amino acid translation for any CDS annotation using the transl_table and codon_start labels on the annotation. This error message occurs when this translation does not start with the start codon, M, and you have not specified that the CDS is partial. To correct this, you will need to change the transl_table and/or codon_start label using the Edit Annotations window so that the first codon is a start codon. Alternatively, if this CDS is a partial CDS sequence, open the Edit Annotations window and double click on the annotation interval. In the window that pops up, specify that the 5′ end of the annotation is truncated. | 移动注释框，使第一位起始密码子编辑氨基酸为M甲硫氨酸。如transl_except:(pos:105458..105460,aa:Met) |
处理方式参照geneious官网
