子实体形态相关基因鉴定

糖类活性酶功能注释

将包含各糖类活性酶家族的隐马尔科夫序列特征谱下载自dbCAN数据库（Yin et al.，2012）。使用Hmmscan软件（Eddy，2009）进行糖类活性酶的注释，在这一过程中，序列特征谱作为搜索的目标，而包含各真菌蛋白质组序列的文件作为搜索对象。生成的初步结果使用dbCAN提供的hmmscan-parser脚本程序进行处理。即 CAZymes

蛋白激酶功能注释

通过Blastp与KinBase（http://kinase.com/kinbase/）数据库进行比对，选用e-value < e-10进行筛选，使用perl软件包对蛋白家族的基因数量进行分类统计，得到FUNCAT数据库的数量矩阵。

KinBase: Kinase Database at Manning's Group

The Mushroom Kinome

• Human: protein, kinase domain, RNA
• Mouse: protein, kinase domain, RNA
• Sea Urchin: protein, kinase domain, RNA
• Fruit Fly: protein, kinase domain, RNA
• Nematode worm: protein, kinase domain, RNA
• A.queenslandica: protein, kinase domain, RNA
• M.brevicollis: protein, kinase domain, RNA
• Bakers Yeast: protein, kinase domain, RNA Saccharomyces cerevisiae
• C.cinerea: protein, kinase domain, RNA Coprinopsis cinerea 下载
• Slime mold: protein, kinase domain, RNA
• Tetrahymena: protein, kinase domain, RNA
• G.lamblia: protein, kinase domain, RNA
• L.major: protein, kinase domain, RNA
• T.vaginalis: protein, kinase domain, RNA
• S.moellendorffii: protein, kinase domain, RNA

# 下载好的KinBase数据库位置如下
/media/aa/DATA/SZQ2/bj_software/KinBase/C.cinerea_kin_dom.fasta
/media/aa/DATA/SZQ2/bj_software/KinBase/C.cinerea_AAprotein.fasta
# 在(pfam_scan)下
conda activate pfam_scan
# 构建数据库
diamond makedb --in /media/aa/DATA/SZQ2/bj_software/KinBase/Ccinerea_kin_dom.fasta -d Ccinerea_kin_dom
# 新建文件夹
mkdir 18.KinBase && cd 18.KinBase
# 1）diamond blastp   evalue为1e-10
for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "diamond blastp --db /media/aa/DATA/SZQ2/bj_software/KinBase/Ccinerea_kin_dom.fasta --query /media/aa/Expansion/szq2/bj/b.OrthoFinder/compliantFasta4/compliantFasta/$i.fasta --out $i.KinBase10.xml --outfmt 5 --sensitive --max-target-seqs 20 --evalue 1e-10 --id 20 --tmpdir /dev/shm --index-chunks 1"
done > command.KinBase.list
ParaFly -c command.KinBase.list -CPU 48
# 新建文件夹
mkdir KinBase10.tab && cd KinBase10.tab
# 2）parsing_blast_result.pl
for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "/media/aa/DATA2/bin/parsing_blast_result.pl --evalue 1e-10 --HSP-num 1 --out-hit-confidence --suject-annotation ../$i.KinBase10.xml > $i.KinBase10.tab"
done > command.KinBase.list
ParaFly -c command.KinBase.list -CPU 48
# 在(jcvi)下
conda activate jcvi
# 3）比对结果中筛选每个query的最佳subject
for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "python -m jcvi.formats.blast best -n 1 $i.KinBase10.tab"
done > command.jcvi.list
ParaFly -c command.jcvi.list -CPU 48
# 4）复制并重命名
mkdir best && cd best
cp ../*.KinBase10.tab.best ./
# 查看每个文件里有多少行，“行数-1”即为注释出给结果总数
wc -l *.KinBase10.tab.best 

微管蛋白功能注释

将包含微管蛋白家族（PF00091.20）和Misato家族（PF10644_misato）种子序列的HMM谱下载自Pfam数据库。使用Hmmsacn软件进行序列的鉴定。初步得到的微管蛋白序列通过系统发育分析方法进行家族分类。

直接从之前的Pfam结果里面筛选。

Pfam: Family: Tubulin (PF00091)

Family: Misat_Tub_SegII (PF10644)

Family: NAD_binding_10 (PF13460)

Family: Tubulin_2 (PF13809)

Family: Tubulin_3 (PF14881)

Family: Tubulin_C (PF03953)

GCP_C_terminal	PF04130.16	Gamma tubulin complex component C-terminal
GCP_N_terminal	PF17681.4	Gamma tubulin complex component N-terminal
MOZART1	PF12554.11	Mitotic-spindle organizing gamma-tubulin ring associated
TBCA	PF02970.19	Tubulin binding cofactor A
TBCC	PF07986.15	Tubulin binding cofactor C
TBCC_N	PF16752.8	Tubulin-specific chaperone C N-terminal domain
TFCD_C	PF12612.11	Tubulin folding cofactor D C terminal
TTL	PF03133.18	Tubulin-tyrosine ligase family
Tubulin	PF00091.28	Tubulin/FtsZ family, GTPase domain
Tubulin_3	PF14881.9	Tubulin domain
Tubulin_C	PF03953.20	Tubulin C-terminal domain
Misat_Tub_SegII	PF10644.12	Misato Segment II tubulin-like domain
NAD_binding_10	PF13460.9	NAD_binding_10

交配型（MAT）基因座的基因组结构分析

基于Non-Redundant Protein Database（NR）（https://www.ncbi.nlm.nih.gov/protein/）、Swiss-Prot（https://www.uniprot.org/）和Pfam（http://pfam.xfam.org/）数据库鉴定同源域转录因子基因（HD）和信息素/受体基因，并通过BLAST搜索进一步鉴定MAT基因座旁的基因。以下序列被用作查询（在NCBI下载）：

用于鉴定线粒体中间肽酶基因（mip）

来自Fomitiporia mediterranea的XP_007265184.1

来自Schizophyllum commune的XP_003038723.1

来自平菇Pleurotus ostreatus的XP_036634433.1

来自双孢蘑菇Agaricus bisporus的XP_007325204.1

来自云芝Trametes versicolor的XP_008032819.1

则用于鉴定β侧翼基因（β-fg）

来自Heterobasidion irregular的XP_009540982.1

来自双孢蘑菇Agaricus bisporus的XP_006454075.1

来自Coprinopsis cinerea的XP_001829147.2

# 下载好的PHI数据库位置如下
/media/aa/DATA/SZQ2/bj_software/MAT/mip/
XP_003038723_1.fasta  XP_007265184_1.fasta  XP_007325204_1.fasta  XP_008032819_1.fasta  XP_036634433_1.fasta
/media/aa/DATA/SZQ2/bj_software/MAT/βfg/
XP_001829147_2.fasta  XP_006454075_1.fasta  XP_009540982_1.fasta
# 在(pfam_scan)下
conda activate pfam_scan
# 构建数据库
diamond makedb --in /media/aa/DATA/SZQ2/bj_software/MAT/mip/XP_003038723_1.fasta -d XP_003038723_1
diamond makedb --in /media/aa/DATA/SZQ2/bj_software/MAT/mip/XP_007265184_1.fasta -d XP_007265184_1
diamond makedb --in /media/aa/DATA/SZQ2/bj_software/MAT/mip/XP_007325204_1.fasta -d XP_007325204_1
diamond makedb --in /media/aa/DATA/SZQ2/bj_software/MAT/mip/XP_008032819_1.fasta -d XP_008032819_1
diamond makedb --in /media/aa/DATA/SZQ2/bj_software/MAT/mip/XP_036634433_1.fasta -d XP_036634433_1
diamond makedb --in /media/aa/DATA/SZQ2/bj_software/MAT/βfg/XP_001829147_2.fasta -d XP_001829147_2
diamond makedb --in /media/aa/DATA/SZQ2/bj_software/MAT/βfg/XP_006454075_1.fasta -d XP_006454075_1
diamond makedb --in /media/aa/DATA/SZQ2/bj_software/MAT/βfg/XP_009540982_1.fasta -d XP_009540982_1
# 新建文件夹
mkdir 20.MAT && cd 20.MAT
# 1）diamond blastp   evalue为1e-10
for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "diamond blastp --db /media/aa/DATA/SZQ2/bj_software/MAT/mip/XP_003038723_1.fasta --query /media/aa/Expansion/szq2/bj/b.OrthoFinder/compliantFasta4/compliantFasta/$i.fasta --out $i.XP_003038723_110.xml --outfmt 5 --sensitive --max-target-seqs 20 --evalue 1e-10 --id 20 --tmpdir /dev/shm --index-chunks 1"
done > command.XP_003038723_1.list
ParaFly -c command.XP_003038723_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "diamond blastp --db /media/aa/DATA/SZQ2/bj_software/MAT/mip/XP_007265184_1.fasta --query /media/aa/Expansion/szq2/bj/b.OrthoFinder/compliantFasta4/compliantFasta/$i.fasta --out $i.XP_007265184_110.xml --outfmt 5 --sensitive --max-target-seqs 20 --evalue 1e-10 --id 20 --tmpdir /dev/shm --index-chunks 1"
done > command.XP_007265184_1.list
ParaFly -c command.XP_007265184_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "diamond blastp --db /media/aa/DATA/SZQ2/bj_software/MAT/mip/XP_007325204_1.fasta --query /media/aa/Expansion/szq2/bj/b.OrthoFinder/compliantFasta4/compliantFasta/$i.fasta --out $i.XP_007325204_110.xml --outfmt 5 --sensitive --max-target-seqs 20 --evalue 1e-10 --id 20 --tmpdir /dev/shm --index-chunks 1"
done > command.XP_007325204_1.list
ParaFly -c command.XP_007325204_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "diamond blastp --db /media/aa/DATA/SZQ2/bj_software/MAT/mip/XP_008032819_1.fasta --query /media/aa/Expansion/szq2/bj/b.OrthoFinder/compliantFasta4/compliantFasta/$i.fasta --out $i.XP_008032819_110.xml --outfmt 5 --sensitive --max-target-seqs 20 --evalue 1e-10 --id 20 --tmpdir /dev/shm --index-chunks 1"
done > command.XP_008032819_1.list
ParaFly -c command.XP_008032819_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "diamond blastp --db /media/aa/DATA/SZQ2/bj_software/MAT/mip/XP_036634433_1.fasta --query /media/aa/Expansion/szq2/bj/b.OrthoFinder/compliantFasta4/compliantFasta/$i.fasta --out $i.XP_036634433_110.xml --outfmt 5 --sensitive --max-target-seqs 20 --evalue 1e-10 --id 20 --tmpdir /dev/shm --index-chunks 1"
done > command.XP_036634433_1.list
ParaFly -c command.XP_036634433_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "diamond blastp --db /media/aa/DATA/SZQ2/bj_software/MAT/βfg/XP_001829147_2.fasta --query /media/aa/Expansion/szq2/bj/b.OrthoFinder/compliantFasta4/compliantFasta/$i.fasta --out $i.XP_001829147_210.xml --outfmt 5 --sensitive --max-target-seqs 20 --evalue 1e-10 --id 20 --tmpdir /dev/shm --index-chunks 1"
done > command.XP_001829147_2.list
ParaFly -c command.XP_001829147_2.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "diamond blastp --db /media/aa/DATA/SZQ2/bj_software/MAT/βfg/XP_006454075_1.fasta --query /media/aa/Expansion/szq2/bj/b.OrthoFinder/compliantFasta4/compliantFasta/$i.fasta --out $i.XP_006454075_110.xml --outfmt 5 --sensitive --max-target-seqs 20 --evalue 1e-10 --id 20 --tmpdir /dev/shm --index-chunks 1"
done > command.XP_006454075_1.list
ParaFly -c command.XP_006454075_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "diamond blastp --db /media/aa/DATA/SZQ2/bj_software/MAT/βfg/XP_009540982_1.fasta --query /media/aa/Expansion/szq2/bj/b.OrthoFinder/compliantFasta4/compliantFasta/$i.fasta --out $i.XP_009540982_110.xml --outfmt 5 --sensitive --max-target-seqs 20 --evalue 1e-10 --id 20 --tmpdir /dev/shm --index-chunks 1"
done > command.XP_009540982_1.list
ParaFly -c command.XP_009540982_1.list -CPU 48

# 新建文件夹
mkdir MAT10.tab && cd MAT10.tab
# 2）parsing_blast_result.pl
for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "/media/aa/DATA2/bin/parsing_blast_result.pl --evalue 1e-10 --HSP-num 1 --out-hit-confidence --suject-annotation ../$i.XP_003038723_110.xml > $i.XP_003038723_110.tab"
done > command.XP_003038723_1.list
ParaFly -c command.XP_003038723_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "/media/aa/DATA2/bin/parsing_blast_result.pl --evalue 1e-10 --HSP-num 1 --out-hit-confidence --suject-annotation ../$i.XP_007265184_110.xml > $i.XP_007265184_110.tab"
done > command.XP_007265184_1.list
ParaFly -c command.XP_007265184_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "/media/aa/DATA2/bin/parsing_blast_result.pl --evalue 1e-10 --HSP-num 1 --out-hit-confidence --suject-annotation ../$i.XP_007325204_110.xml > $i.XP_007325204_110.tab"
done > command.XP_007325204_1.list
ParaFly -c command.XP_007325204_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "/media/aa/DATA2/bin/parsing_blast_result.pl --evalue 1e-10 --HSP-num 1 --out-hit-confidence --suject-annotation ../$i.XP_008032819_110.xml > $i.XP_008032819_110.tab"
done > command.XP_008032819_1.list
ParaFly -c command.XP_008032819_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "/media/aa/DATA2/bin/parsing_blast_result.pl --evalue 1e-10 --HSP-num 1 --out-hit-confidence --suject-annotation ../$i.XP_036634433_110.xml > $i.XP_036634433_110.tab"
done > command.XP_036634433_1.list
ParaFly -c command.XP_036634433_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "/media/aa/DATA2/bin/parsing_blast_result.pl --evalue 1e-10 --HSP-num 1 --out-hit-confidence --suject-annotation ../$i.XP_001829147_210.xml > $i.XP_001829147_210.tab"
done > command.XP_001829147_2.list
ParaFly -c command.XP_001829147_2.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "/media/aa/DATA2/bin/parsing_blast_result.pl --evalue 1e-10 --HSP-num 1 --out-hit-confidence --suject-annotation ../$i.XP_006454075_110.xml > $i.XP_006454075_110.tab"
done > command.XP_006454075_1.list
ParaFly -c command.XP_006454075_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "/media/aa/DATA2/bin/parsing_blast_result.pl --evalue 1e-10 --HSP-num 1 --out-hit-confidence --suject-annotation ../$i.XP_009540982_110.xml > $i.XP_009540982_110.tab"
done > command.XP_009540982_1.list
ParaFly -c command.XP_009540982_1.list -CPU 48

# 在(jcvi)下
conda activate jcvi
# 3）比对结果中筛选每个query的最佳subject
for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "python -m jcvi.formats.blast best -n 1 $i.XP_003038723_110.tab"
done > command.jcviXP_003038723_1.list
ParaFly -c command.jcviXP_003038723_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "python -m jcvi.formats.blast best -n 1 $i.XP_007265184_110.tab"
done > command.jcviXP_007265184_1.list
ParaFly -c command.jcviXP_007265184_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "python -m jcvi.formats.blast best -n 1 $i.XP_007325204_110.tab"
done > command.jcviXP_007325204_1.list
ParaFly -c command.jcviXP_007325204_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "python -m jcvi.formats.blast best -n 1 $i.XP_008032819_110.tab"
done > command.jcviXP_008032819_1.list
ParaFly -c command.jcviXP_008032819_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "python -m jcvi.formats.blast best -n 1 $i.XP_036634433_110.tab"
done > command.jcviXP_036634433_1.list
ParaFly -c command.jcviXP_036634433_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "python -m jcvi.formats.blast best -n 1 $i.XP_001829147_210.tab"
done > command.jcviXP_001829147_2.list
ParaFly -c command.jcviXP_001829147_2.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "python -m jcvi.formats.blast best -n 1 $i.XP_006454075_110.tab"
done > command.jcviXP_006454075_1.list
ParaFly -c command.jcviXP_006454075_1.list -CPU 48

for i in `cat /media/aa/DATA/SZQ2/bj/functional_annotation/94listssp.txt`
do
    echo "python -m jcvi.formats.blast best -n 1 $i.XP_009540982_110.tab"
done > command.jcviXP_009540982_1.list
ParaFly -c command.jcviXP_009540982_1.list -CPU 48
# 4）复制并重命名
mkdir best && cd best
cp ../*.tab.best ./
# 查看每个文件里有多少行，“行数-1”即为注释出给结果总数
wc -l *.XP_003038723_110.tab.best
wc -l *.XP_007265184_110.tab.best
wc -l *.XP_007325204_110.tab.best
wc -l *.XP_008032819_110.tab.best
wc -l *.XP_036634433_110.tab.best
wc -l *.XP_001829147_210.tab.best
wc -l *.XP_006454075_110.tab.best
wc -l *.XP_009540982_110.tab.best

 统计数据