宏基因组实战之：基因预测

紧接前文，各个样本序列组装完毕后，下一步自然是想要弄清楚组装出来的序列中到底是些啥子东西嘛。基因预测方法和工具见这篇推文。

注释工具

常见原核生物基因注释包括 prokka、Prodigal、GeneMark等工具。

这里使用Prodigal对组装的基因组序列进行基因预测：

Prodigal软件参数

-------------------------------------
PRODIGAL v2.6.3 [February, 2016]         
Univ of Tenn / Oak Ridge National Lab
Doug Hyatt, Loren Hauser, et al.     
-------------------------------------

Usage:  prodigal [-a trans_file] [-c] [-d nuc_file] [-f output_type]
                 [-g tr_table] [-h] [-i input_file] [-m] [-n] [-o output_file]
                 [-p mode] [-q] [-s start_file] [-t training_file] [-v]

         -a:  Write protein translations to the selected file.
         -c:  Closed ends.  Do not allow genes to run off edges.
         -d:  Write nucleotide sequences of genes to the selected file.
         -f:  Select output format (gbk, gff, or sco).  Default is gbk.
         -g:  Specify a translation table to use (default 11).
         -h:  Print help menu and exit.
         -i:  Specify FASTA/Genbank input file (default reads from stdin).
         -m:  Treat runs of N as masked sequence; don't build genes across them.
         -n:  Bypass Shine-Dalgarno trainer and force a full motif scan.
         -o:  Specify output file (default writes to stdout).
         -p:  Select procedure (single or meta).  Default is single.
         -q:  Run quietly (suppress normal stderr output).
         -s:  Write all potential genes (with scores) to the selected file.
         -t:  Write a training file (if none exists); otherwise, read and use
              the specified training file.
         -v:  Print version number and exit.

注释参数

prodigal -i final.contigs.fa  # 输入组装后的序列文件
	-a protein.fa             # 输出预测的蛋白序列
	-d gene.fa                # 输数预测的基因序列
	-p meta                   # 默认单菌样本(single)，这里是宏基因组样本
	-f gff 					  # 输出注释文件格式为gff
	-o contigs.gff 			  # 输出注释结果文件
	-s contigs.stat			  # 注释结果文件统计信息

结果文件

预测基因结果：

预测的蛋白结果：

预测结果gff文件：

预测结果统计信息：



这里再使用Prokka预测看下结果如何

Prokka 软件参数

软件参数，太长了收起来，点击展开

Name:
  Prokka 1.12 by Torsten Seemann <torsten.seemann@gmail.com>
Synopsis:
  rapid bacterial genome annotation
Usage:
  prokka [options] <contigs.fasta>
General:
  --help            This help
  --version         Print version and exit
  --docs            Show full manual/documentation
  --citation        Print citation for referencing Prokka
  --quiet           No screen output (default OFF)
  --debug           Debug mode: keep all temporary files (default OFF)
Setup:
  --listdb          List all configured databases
  --setupdb         Index all installed databases
  --cleandb         Remove all database indices
  --depends         List all software dependencies
Outputs:
  --outdir [X]      Output folder [auto] (default '')
  --force           Force overwriting existing output folder (default OFF)
  --prefix [X]      Filename output prefix [auto] (default '')
  --addgenes        Add 'gene' features for each 'CDS' feature (default OFF)
  --addmrna         Add 'mRNA' features for each 'CDS' feature (default OFF)
  --locustag [X]    Locus tag prefix [auto] (default '')
  --increment [N]   Locus tag counter increment (default '1')
  --gffver [N]      GFF version (default '3')
  --compliant       Force Genbank/ENA/DDJB compliance: --addgenes --mincontiglen 200 --centre XXX (default OFF)
  --centre [X]      Sequencing centre ID. (default '')
  --accver [N]      Version to put in Genbank file (default '1')
Organism details:
  --genus [X]       Genus name (default 'Genus')
  --species [X]     Species name (default 'species')
  --strain [X]      Strain name (default 'strain')
  --plasmid [X]     Plasmid name or identifier (default '')
Annotations:
  --kingdom [X]     Annotation mode: Archaea|Bacteria|Mitochondria|Viruses (default 'Bacteria')
  --gcode [N]       Genetic code / Translation table (set if --kingdom is set) (default '0')
  --gram [X]        Gram: -/neg +/pos (default '')
  --usegenus        Use genus-specific BLAST databases (needs --genus) (default OFF)
  --proteins [X]    FASTA or GBK file to use as 1st priority (default '')
  --hmms [X]        Trusted HMM to first annotate from (default '')
  --metagenome      Improve gene predictions for highly fragmented genomes (default OFF)
  --rawproduct      Do not clean up /product annotation (default OFF)
  --cdsrnaolap      Allow [tr]RNA to overlap CDS (default OFF)
Computation:
  --cpus [N]        Number of CPUs to use [0=all] (default '8')
  --fast            Fast mode - only use basic BLASTP databases (default OFF)
  --noanno          For CDS just set /product="unannotated protein" (default OFF)
  --mincontiglen [N] Minimum contig size [NCBI needs 200] (default '1')
  --evalue [n.n]    Similarity e-value cut-off (default '1e-06')
  --rfam            Enable searching for ncRNAs with Infernal+Rfam (SLOW!) (default '0')
  --norrna          Don't run rRNA search (default OFF)
  --notrna          Don't run tRNA search (default OFF)
  --rnammer         Prefer RNAmmer over Barrnap for rRNA prediction (default OFF)

使用前还需安装些perl依赖：cpanm Bio::Perl XML::Simple
prokka --listdb 查看数据库信息

注释参数

prokka --metagenome --prefix prokka --outdir prokka_res final.contigs.fa