【佳学基因检测】基因测序结果如何通过GENECODE/CCDS进行解码检测

当前版本的 ANNOVAR 没有为 GENCODE 提供特定的关键字，但 ANNOVAR 的通用性足以处理 GENCODE 或任何其他基因定义。 GENCODE 系统在过去几年中发生了很大变化，最新版本是 2014 年 2 月的 V19。在 V19 中，BASIC 轨道现已可用，其中包含基于以下描述的高质量基因定义：“GENCODE Basic 该集旨在提供对大多数用户有用的 GENCODE 转录本注释的简化子集。目标是拥有一个覆盖所有基因座的高质量基本集。选择 GENCODE 注释以包含在基本集中独立确定每个基因位点的编码和非编码转录本。”

下面是基因解码可以用于变异注释的 GENCODE 基因定义的命令（请注意，如果 humandb/hg19_seq 中没有全基因组 FASTA 文件，基因检测与基因测序机构应该首先执行 annotate_variation.pl -downdb -build hg19 seq humandb/hg19_seq/ ）。

[jiaxuegene@genejiedu ~/]$ annotate_variation.pl -downdb wgEncodeGencodeBasicV19 humandb/ -build hg19
NOTICE: Web-based checking to see whether ANNOVAR new version is available ... Done
NOTICE: Downloading annotation database http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/wgEncodeGencodeBasicV19.txt.gz ... OK
NOTICE: Uncompressing downloaded files
NOTICE: Finished downloading annotation files for hg19 build version, with files saved at the 'humandb' directory

[jiaxuegene@genejiedu ~/]$ retrieve_seq_from_fasta.pl -format genericGene -seqdir humandb/hg19_seq/ -outfile humandb/hg19_wgEncodeGencodeBasicV19Mrna.fa humandb/hg19_wgEncodeGencodeBasicV19.txt 

[jiaxuegene@genejiedu ~/]$ annotate_variation.pl -build hg19 -out ex1 -dbtype wgEncodeGencodeBasicV19 example/ex1.avinput humandb/ 
NOTICE: The --geneanno operation is set to ON by default
NOTICE: Reading gene annotation from humandb/hg19_wgEncodeGencodeBasicV19.txt ... Done with 95929 transcripts (including 38291 without coding sequence annotation) for 42594 unique genes
NOTICE: Reading FASTA sequences from humandb/hg19_wgEncodeGencodeBasicV19Mrna.fa ... Done with 16 sequences
WARNING: A total of 303 sequences will be ignored due to lack of correct ORF annotation
NOTICE: Finished gene-based annotation on 15 genetic variants in example/ex1.avinput
NOTICE: Output files were written to ex1.variant_function, ex1.exonic_variant_function
Then check the results below:

[[jiaxuegene@genejiedu ~/]$ cat ex1.variant_function
UTR5 ISG15(ENST00000379389.4:c.-33T>C) 1 948921 948921 T C comments: rs15842, a SNP in 5' UTR of ISG15
UTR3 ATAD3C(ENST00000378785.2:c.*91G>T) 1 1404001 1404001 G T comments: rs149123833, a SNP in 3' UTR of ATAD3C
splicing NPHP4(ENST00000378156.4:exon22:c.2818-2T>A) 1 5935162 5935162 A T comments: rs1287637, a splice site variant in NPHP4
intronic DDR2 1 162736463 162736463 C T comments: rs1000050, a SNP in Illumina SNP arrays
intronic DNASE2B 1 84875173 84875173 C T comments: rs6576700 or SNP_A-1780419, a SNP in Affymetrix SNP arrays
intergenic RP13-221M14.3(dist=46825),PRAMEF26(dist=5062) 1 13211293 13211294 TC - comments: rs59770105, a 2-bp deletion
intergenic UBIAD1(dist=48375),PTCHD2(dist=135699) 1 11403596 11403596 - AT comments: rs35561142, a 2-bp insertion
intergenic RP11-364B6.1(dist=872522),RP11-251P6.1(dist=652543) 1 105492231 105492231 A ATAAA comments: rs10552169, a block substitution
exonic IL23R 1 67705958 67705958 G A comments: rs11209026 (R381Q), a SNP in IL23R associated with Crohn's disease
exonic ATG16L1 2 234183368 234183368 A G comments: rs2241880 (T300A), a SNP in the ATG16L1 associated with Crohn's disease
exonic NOD2 16 50745926 50745926 C T comments: rs2066844 (R702W), a non-synonymous SNP in NOD2
exonic NOD2 16 50756540 50756540 G C comments: rs2066845 (G908R), a non-synonymous SNP in NOD2
exonic NOD2 16 50763778 50763778 - C comments: rs2066847 (c.3016_3017insC), a frameshift SNP in NOD2
exonic GJB2 13 20763686 20763686 G - comments: rs1801002 (del35G), a frameshift mutation in GJB2, associated with hearing loss
exonic CRYL1,GJB6 13 20797176 21105944 0 - comments: a 342kb deletion encompassing GJB6, associated with hearing loss

[jiaxuegene@genejiedu ~/]$ cat ex1.exonic_variant_function
line9 nonsynonymous SNV IL23R:ENST00000395227.1:exon4:c.G377A:p.R126Q,IL23R:ENST00000347310.5:exon9:c.G1142A:p.R381Q, 1 67705958 6770595comments: rs11209026 (R381Q), a SNP in IL23R associated with Crohn's disease
line10 nonsynonymous SNV ATG16L1:ENST00000373525.5:exon6:c.A466G:p.T156A,ATG16L1:ENST00000347464.5:exon5:c.A409G:p.T137A,ATG16L1:ENST00000392018.1:exon10:c.A949G:p.T317A,ATG16L1:ENST00000392017.4:exon9:c.A898G:p.T300A,ATG16L1:ENST00000392020.4:exon8:c.A841G:p.T281A, 2 234183368 234183368 comments: rs2241880 (T300A), a SNP in the ATG16L1 associated with Crohn's disease
line11 nonsynonymous SNV NOD2:ENST00000300589.2:exon4:c.C2104T:p.R702W, 16 50745926 50745926 C T comments: rs2066844 (R702W), a non-synonymous SNP in NOD2
line12 nonsynonymous SNV NOD2:ENST00000300589.2:exon8:c.G2722C:p.G908R, 16 50756540 50756540 G C comments: rs2066845 (G908R), a non-synonymous SNP in NOD2
line13 frameshift insertion NOD2:ENST00000300589.2:exon11:c.3017dupC:p.A1006fs, 16 50763778 50763778 - C comments: rs2066847 (c.3016_3017insC), a frameshift SNP in NOD2
line14 frameshift deletion GJB2:ENST00000382848.4:exon2:c.35delG:p.G12fs,GJB2:ENST00000382844.1:exon1:c.35delG:p.G12fs, 13 20763686 2076368comments: rs1801002 (del35G), a frameshift mutation in GJB2, associated with hearing loss
line15 frameshift deletion GJB6:ENST00000241124.6:wholegene,CRYL1:ENST00000298248.7:wholegene,GJB6:ENST00000400065.3:wholegene,CRYL1:ENST00000382812.1:wholegene,GJB6:ENST00000356192.6:wholegene,GJB6:ENST00000400066.3:wholegene, 13 20797176 21105944 0 - comments: a 342kb deletion encompassing GJB6, associated with hearing loss

同样，基因解码可以使用不同的 -dbtype 参数（wgEncodeGencodeCompV19 和 wgEncodeGencodePolyaV19）切换到 GENCODE 的综合注释和 PolyA 注释。

使用这些注释时，务必注意要使用的正确文件名（如果您不确定表名，请使用 UCSC 基因组浏览器中的表浏览器）。

还支持许多其他基因定义系统。例如，用户可以选择CCDS基因定义。（请注意，如果 humandb/hg19_seq 中没有全基因组 FASTA 文件，获得基因序列后应该首先执行 annotate_variation.pl -downdb -build hg19 seq humandb/hg19_seq/）。

[jiaxuegene@genejiedu ~/]$ annotate_variation.pl -downdb -build hg19 ccdsGene humandb

[jiaxuegene@genejiedu ~/]$ retrieve_seq_from_fasta.pl humandb/hg19_ccdsGene.txt -seqdir humandb/hg19_seq -format refGene -outfile humandb/hg19_ccdsGeneMrna.fa

[jiaxuegene@genejiedu ~/]$ annotate_variation.pl -buildver hg19 -out ex1 -dbtype ccdsGene example/ex1.avinput humandb/
NOTICE: The --geneanno operation is set to ON by default
NOTICE: Reading gene annotation from humandb/hg19_ccdsGene.txt ... Done with 29045 transcripts (including 0 without coding sequence annotation) for 29014 unique genes
NOTICE: Reading FASTA sequences from humandb/hg19_ccdsGeneMrna.fa ... Done with 8 sequences
WARNING: A total of 38 sequences will be ignored due to lack of correct ORF annotation
NOTICE: Finished gene-based annotation on 15 genetic variants in example/ex1.avinput
NOTICE: Output files were written to ex1.variant_function, ex1.exonic_variant_functiona

基因解码技术解释：对于 CCDS 基因，输出将不包含基因名称，而仅包含 CCDS 标识符。要获取基因名称，基因检测机构必须编写自己的程序来处理 ANNOVAR 输出文件。有两个表可用于将 CCDS ID 转换为其他 ID：ccdsNotes 将 CCDS 转换为 UCSC 已知基因转录本 ID，然后您可以将其转换为基因名称。 ccdsInfo 表将 CCDS ID 转换为 ENSEMBL 转录本或 RefSeq 转录本，然后您可以进一步将它们转换为基因名称。

对基因测序结果进行解读检测通常会涉及以下几个步骤:

1. 将测序reads对参考基因组进行mapping,得到read alignments。常用的工具包括BWA,Bowtie2等。

2. 根据参考基因组的注释信息(例如GENCODE/CCDS),将read alignments与基因、转录本进行比较,看reads是否可map到已知的基因区域。这可以使用一些visual化浏览器进行,如IGV。

3. 对于未map到已知基因区域的reads,可以进行de novo组装,组装成未知的转录本序列。这可以用一些软件工具完成,如Trinity,SOAPdenovo-Trans等。

4. 将组装得到的转录本与已知蛋白数据库(如UniProt)进行序列比对,判断转录本是否编码未知蛋白。

5. 对coding potential较高的未知转录本,可以进行进一步的功能预测分析,如同源比对判断家族,结构预测等。也可以进行实验验证是否真的表达。

6. 最终,整合已知基因注释和新发现的转录本信息,完成序列测序分析和检测。可以确定已知基因的表达水平变化,也可以发现编码潜在新蛋白的未知转录本。

综上,利用已有的基因注释数据库(如GENCODE/CCDS)作为参考,结合reads mapping、de novo组装和功能预测等分析,可以较全面地解析和检测基因测序结果。

(责任编辑：佳学基因)