一、NCBI blast+

1. 安装配置BLAST+程序

在ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/中下载最新的BLAST可执行程序（不要下载源代码`，源码编译非常慢），选择预编译版本，如ncbi-blast-2.2.30+-x64-linux.tar.gz。如果服务器能联网，可直接用wget下载。或者，下载后用SFTP客户端传输到服务器上。

wget ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/ncbi-blast-2.2.30+-x64-linux.tar.gz

解压缩：

tar -zxvf ncbi-blast-2.2.30+-x64-linux.tar.gz

2.基本用法

**提示：blast输出格式有多种，其中11包含信息最全，其它格式都可用blast_formatter程序由11转化为其它格式。所以，比对结果请使用11格式。

1) 对相应的序列进行建库

makeblastdb -in db.fasta -dbtype nucl -parse_seqids -out dbname

**其中 -dbtype 为 nucl 则表示对核酸类型的序列建库，为 prot 则表示对氨基酸类型的序列进行建库

2) 建库之后，就是拿目标序列比对

blastn -query test.fa -db daname -outfmt 11 -out "test.blastn@nr.asn" -num_threads 8

**其中输出文件名test.blastn@nr.asn是个人习惯，即“序列文件名.blast子程序名@库名.结果格式”，结果简单明了

**如果目标序列是蛋白序列，匹配到 nr 数据库或者其他蛋白类数据库，以及其他自己构建的蛋白序列库时，则用 blastp, 其他参数类似。

二、diamond程序

1. 安装diamond程序

在diamond下载界面获得下载链接

wget http://github.com/bbuchfink/diamond/releases/download/v0.9.17/diamond-linux64.tar.gz

tar xzf diamond-linux64.tar.gz

**解压结果为一个二进制可执行文件 diamond, 直接添加环境变量即可

2. 基本用法

To now run an alignment task, we assume to have a protein database file in FASTA format named nr.faa and a file of DNA reads that we want to align namedreads.fna.

1) 建库 In order to set up a reference database for DIAMOND, the makedb command needs to be executed with the following command line:

$ diamond makedb --in nr.faa -d nr ## 建库

$ diamond help

diamond helpdiamond v0.8.8.70 | by Benjamin BuchfinkCheck http://github.com/bbuchfink/diamond for updates.

Syntax: diamond COMMAND [OPTIONS]

Commands:

makedb Build DIAMOND database from a FASTA file

blastp Align amino acid query sequences against a protein reference database

blastx Align DNA query sequences against a protein reference database

view View DIAMOND alignment archive (DAA) formatted file

help Produce help message

version Display version information

General options:

--threads (-p)        number of CPU threads

--db (-d)              database file

--daa (-a)            DIAMOND alignment archive (DAA) file

--verbose (-v)        verbose console output

--log                  enable debug log

--quiet                disable console output

Makedb options:

--in                  input reference file in FASTA format

--block-size (-b)      sequence block size in billions of letters (default=2)

Aligner options:

--query (-q)          input query file

--max-target-seqs (-k) maximum number of target sequences to report alignments for

--top                  report alignments within this percentage range of top alignment score (overrides --max-target-seqs)

--compress            compression for output files (0=none, 1=gzip)

--evalue (-e)          maximum e-value to report alignments

--min-score            minimum bit score to report alignments (overrides e-value setting)

--id                  minimum identity% to report an alignment

--query-cover          minimum query cover% to report an alignment

--sensitive            enable sensitive mode (default: fast)

--index-chunks (-c)    number of chunks for index processing

--tmpdir (-t)          directory for temporary files

--gapopen              gap open penalty (default=11 for protein)

--gapextend            gap extension penalty (default=1 for protein)

--matrix              score matrix for protein alignment

--seg                  enable SEG masking of queries (yes/no)

--salltitles          print full subject titles in output files

Advanced options:

--seed-freq            maximum seed frequency

--run-len (-l)        mask runs between stop codons shorter than this length

--max-hits (-C)        maximum number of hits to consider for one seed

--id2                  minimum number of identities for stage 1 hit

--window (-w)          window size for local hit search

--xdrop (-x)          xdrop for ungapped alignment

--gapped-xdrop (-X)    xdrop for gapped alignment in bits

--ungapped-score      minimum raw alignment score to continue local extension

--hit-band            band for hit verification

--hit-score            minimum score to keep a tentative alignment

--band                band for dynamic programming computation

--shapes (-s)          number of seed shapes (0 = all available)

--index-mode          index mode (0=4x12, 1=16x9)

--fetch-size          trace point fetch size

--single-domain        Discard secondary domains within one target sequence

--dbsize              effective database size (in letters)

--no-auto-append      disable auto appending of DAA and DMND file extensions

View options:

--out (-o)            output file

--outfmt (-f)          output format (tab/sam/xml)

--forwardonly          only show alignments of forward strand

2) 序列比对

** 上面建库之后会生成一个 nr.dmnd 文件，The alignment task may then be initiated using the blastx command like this:

$ diamond blastx -d nr -q reads.fna -o matches.m8

The output file here is specified with the –o option and named matches.m8. By default, it is generated in BLAST tabular format.