【叶绿体基因组】注释校正与tbl文件的生成

叶绿体基因组注释是非常重要的一步，注释的合理与否直接影响后续分析的正确性，例如密码子偏好性分析、选择压力分析等等。

STEP 1 获取初始的GB注释文件

使用GeSeq进行注释。MPI-MP CHLOROBOX - GeSeq (mpg.de)
当然，其他软件例如CPGAVAS2也可以进行注释。
这里以Tetrastigma hemsleyanum（三叶青，三叶崖爬藤）为例。其他参数配置可以参考以下选择。

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STEP 2 注释校正（一）

通常来说，得到的注释文件不能直接使用，需要手动调整。
由于注释器的选择、Blast数据库等等因素，初次注释得到的结果中很可能出现以下情况：

重复

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方向不一致
SSC的构象相反了。严格来说，SSC并没有固定的方向，即两种构象的是对的，但是为了后续分析的一致性，这种情况需要被重视。

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内部含有终止密码子（图中的*号）

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可以根据以下几点来选择更为合理的注释：

CDS碱基数目通常为3的倍数。
CDS一般以起始蛋白M开头。但也存在例外，例如ndhD。
根据其他的可信度高的注释结果进行判断。
对于SSC方向不一致的序列，可以先找到两个IR区域和SSC的边界，然后选择SSC的序列，进行反向互补操作，再重新注释。

STEP 3 注释校正（二）

进入GB2SequinMPI-MP CHLOROBOX - GB2sequin (mpg.de)

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选择相应的按钮。点击上传，在右侧的选项中勾选“Complete”“Circular”，使用细菌、古菌和植物质体的密码子表，Location选择chloroplast，即叶绿体，勾选最下方复选框后，点击“Start Conversion”。

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3.运行结束后，可以在右下方的Results中预览，点击Validation即可看到更加详细的内容。这里我直接分析了Geseq生成的gb文件，而不是严格的ncbi格式的gb文件，所以前面跳了很多未知限定符，比如“info”“annotator”，但是这个不重要，GB2Sequin会自动删除。

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除了上面提到的位置限定符报错，其他的就是需要我们仔细查看的报错或警告了。

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点击Validation，这个实际上就是一个txt文件。

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ERROR: valid [SEQ_FEAT.StartCodon] Illegal start codon used. Wrong genetic code [11] or protein should be partial FEATURE: CDS: photosystem II subunit L [lcl|NC_029339.:c67971-67855] [lcl|NC_029339.: raw, dna len= 159889] -> [lcl|NC_029339._42]
ERROR: valid [SEQ_FEAT.StartCodon] Illegal start codon used. Wrong genetic code [11] or protein should be partial FEATURE: CDS: NADH dehydrogenase subunit D [lcl|NC_029339.:c122084-120570] [lcl|NC_029339.: raw, dna len= 159889] -> [lcl|NC_029339._79]
ERROR: valid [SEQ_FEAT.NoProtein] No protein Bioseq given FEATURE: CDS:  [lcl|NC_029339.:159794-159889] [lcl|NC_029339.: raw, dna len= 159889]
ERROR: valid [SEQ_FEAT.NoStop] Missing stop codon FEATURE: CDS:  [lcl|NC_029339.:159794-159889] [lcl|NC_029339.: raw, dna len= 159889]
ERROR: valid [SEQ_FEAT.MissingCDSproduct] Expected CDS product absent FEATURE: CDS:  [lcl|NC_029339.:159794-159889] [lcl|NC_029339.: raw, dna len= 159889]
WARNING: valid [SEQ_FEAT.CollidingGeneNames] Colliding names in gene features FEATURE: Gene: accD [lcl|NC_029339.:61229-62797] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: ndhB [lcl|NC_029339.:146828-149044] [lcl|NC_029339.: raw, dna len= 159889]
WARNING: valid [SEQ_FEAT.CollidingGeneNames] Colliding names in gene features FEATURE: Gene: ndhK [lcl|NC_029339.:c53866-53120] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: rpl2 [lcl|NC_029339.:158238-159727] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: rpl23 [lcl|NC_029339.:157938-158219] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: rps12 [(lcl|NC_029339.:c73732-73619, 145193-145986)] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: rps7 [lcl|NC_029339.:146040-146507] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: rrn16 [lcl|NC_029339.:c143071-141581] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: rrn23 [lcl|NC_029339.:c139186-136375] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: rrn4.5 [lcl|NC_029339.:c136277-136175] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: rrn5 [lcl|NC_029339.:c135959-135839] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: trnA-UGC [lcl|NC_029339.:c140206-139344] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: trnI-CAU [lcl|NC_029339.:157699-157772] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: trnI-GAU [lcl|NC_029339.:c141280-140271] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: trnL-CAA [lcl|NC_029339.:149623-149703] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: trnN-GUU [lcl|NC_029339.:134835-134906] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: trnR-ACG [lcl|NC_029339.:c135577-135504] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: trnV-GAC [lcl|NC_029339.:c143362-143291] [lcl|NC_029339.: raw, dna len= 159889]
WARNING: valid [SEQ_FEAT.CollidingGeneNames] Colliding names in gene features FEATURE: Gene: ycf1 [lcl|NC_029339.:c134518-128885] [lcl|NC_029339.: raw, dna len= 159889]
NOTE: valid [SEQ_FEAT.ReplicatedGeneSequence] Colliding names in gene features, but underlying sequences are identical FEATURE: Gene: ycf2 [lcl|NC_029339.:c157610-150717] [lcl|NC_029339.: raw, dna len= 159889]
WARNING: valid [SEQ_FEAT.GeneXrefNeeded] Feature overlapped by 2 identical-length equivalent genes but has no cross-reference FEATURE: exon: /number=1 [lcl|NC_029339.:c73732-73619] [lcl|NC_029339.: raw, dna len= 159889]
ERROR: valid [SEQ_INST.BadProteinStart] gap symbol at start of protein sequence (psbL - photosystem II subunit L) BIOSEQ: lcl|NC_029339._42: raw, aa len= 38
ERROR: valid [SEQ_INST.BadProteinStart] gap symbol at start of protein sequence (ndhD - NADH dehydrogenase subunit D) BIOSEQ: lcl|NC_029339._79: raw, aa len= 504

从结果可以看出，总共7个ERROR，4个WARNING。其中

SEQ_FEAT.StartCodon  起始密码子
SEQ_INST.BadProteinStart  起始蛋白
SEQ_FEAT.NoProtein  蛋白序列缺失
SEQ_FEAT.NoStop  终止密码子缺失
SEQ_FEAT.MissingCDSproduct  编码产物缺失
SEQ_FEAT.CollidingGeneNames  基因名称冲突

第一个和第二个ERROR都提示使用了错误的起始密码子，这也和最后两个ERROR有关。它们的位置在c67971-67855和c122084-120570，分别是psbL和ndhD。在不能确定是否正确时，我们可以根据其他研究的注释结果来确定，这一次的psbL和ndhD的注释结果和先前的研究吻合，暂时可以使用。这一项报错就不需要管了。
其他情况类似，只需要仔细查看Validation.txt并且修改注释信息即可。需要注意的是，在修改注释时，需要保持一致性，例如修改了某个trna的产物，只改了trna，而没有改对应的gene，也是会报错的。
每次修改完后，再导入到GB2Sequin进行分析，如此往复，即可完成校正。

STEP 4 tbl文件生成

点击Annotation Table，点击弹窗的Download，即可下载。tbl文件用于将序列上传至ncbi。