【佳學(xué)基因檢測(cè)】基因測(cè)序結(jié)果如何通過(guò)GENECODE/CCDS進(jìn)行解碼檢測(cè)

當(dāng)前版本的 ANNOVAR 沒(méi)有為 GENCODE 提供特定的關(guān)鍵字，但 ANNOVAR 的通用性足以處理 GENCODE 或任何其他基因定義。 GENCODE 系統(tǒng)在過(guò)去幾年中發(fā)生了很大變化，賊新版本是 2014 年 2 月的 V19。在 V19 中，BASIC 軌道現(xiàn)已可用，其中包含基于以下描述的高質(zhì)量基因定義：“GENCODE Basic 該集旨在提供對(duì)大多數(shù)用戶有用的 GENCODE 轉(zhuǎn)錄本注釋的簡(jiǎn)化子集。目標(biāo)是擁有一個(gè)覆蓋所有基因座的高質(zhì)量基本集。選擇 GENCODE 注釋以包含在基本集中獨(dú)立確定每個(gè)基因位點(diǎn)的編碼和非編碼轉(zhuǎn)錄本。”

下面是基因解碼可以用于變異注釋的 GENCODE 基因定義的命令（請(qǐng)注意，如果 humandb/hg19_seq 中沒(méi)有全基因組 FASTA 文件，基因檢測(cè)與基因測(cè)序機(jī)構(gòu)應(yīng)該首先執(zhí)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

同樣，基因解碼可以使用不同的 -dbtype 參數(shù)（wgEncodeGencodeCompV19 和 wgEncodeGencodePolyaV19）切換到 GENCODE 的綜合注釋和 PolyA 注釋。

使用這些注釋時(shí)，務(wù)必注意要使用的正確文件名（如果您不確定表名，請(qǐng)使用 UCSC 基因組瀏覽器中的表瀏覽器）。

還支持許多其他基因定義系統(tǒng)。例如，用戶可以選擇CCDS基因定義。（請(qǐng)注意，如果 humandb/hg19_seq 中沒(méi)有全基因組 FASTA 文件，獲得基因序列后應(yīng)該首先執(zhí)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

基因解碼技術(shù)解釋：對(duì)于 CCDS 基因，輸出將不包含基因名稱，而僅包含 CCDS 標(biāo)識(shí)符。要獲取基因名稱，基因檢測(cè)機(jī)構(gòu)必須編寫自己的程序來(lái)處理 ANNOVAR 輸出文件。有兩個(gè)表可用于將 CCDS ID 轉(zhuǎn)換為其他 ID：ccdsNotes 將 CCDS 轉(zhuǎn)換為 UCSC 已知基因轉(zhuǎn)錄本 ID，然后您可以將其轉(zhuǎn)換為基因名稱。 ccdsInfo 表將 CCDS ID 轉(zhuǎn)換為 ENSEMBL 轉(zhuǎn)錄本或 RefSeq 轉(zhuǎn)錄本，然后您可以進(jìn)一步將它們轉(zhuǎn)換為基因名稱。

對(duì)基因測(cè)序結(jié)果進(jìn)行解讀檢測(cè)通常會(huì)涉及以下幾個(gè)步驟:

1. 將測(cè)序reads對(duì)參考基因組進(jìn)行mapping,得到read alignments。常用的工具包括BWA,Bowtie2等。

2. 根據(jù)參考基因組的注釋信息(例如GENCODE/CCDS),將read alignments與基因、轉(zhuǎn)錄本進(jìn)行比較,看reads是否可map到已知的基因區(qū)域。這可以使用一些visual化瀏覽器進(jìn)行,如IGV。

3. 對(duì)于未map到已知基因區(qū)域的reads,可以進(jìn)行de novo組裝,組裝成未知的轉(zhuǎn)錄本序列。這可以用一些軟件工具完成,如Trinity,SOAPdenovo-Trans等。

4. 將組裝得到的轉(zhuǎn)錄本與已知蛋白數(shù)據(jù)庫(kù)(如UniProt)進(jìn)行序列比對(duì),判斷轉(zhuǎn)錄本是否編碼未知蛋白。

5. 對(duì)coding potential較高的未知轉(zhuǎn)錄本,可以進(jìn)行進(jìn)一步的功能預(yù)測(cè)分析,如同源比對(duì)判斷家族,結(jié)構(gòu)預(yù)測(cè)等。也可以進(jìn)行實(shí)驗(yàn)驗(yàn)證是否真的表達(dá)。

6. 賊終,整合已知基因注釋和新發(fā)現(xiàn)的轉(zhuǎn)錄本信息,完成序列測(cè)序分析和檢測(cè)?？梢源_定已知基因的表達(dá)水平變化,也可以發(fā)現(xiàn)編碼潛在新蛋白的未知轉(zhuǎn)錄本。

綜上,利用已有的基因注釋數(shù)據(jù)庫(kù)(如GENCODE/CCDS)作為參考,結(jié)合reads mapping、de novo組裝和功能預(yù)測(cè)等分析,可以較全面地解析和檢測(cè)基因測(cè)序結(jié)果。

(責(zé)任編輯：佳學(xué)基因)