導(dǎo)讀
用query基因誓琼、DNA序列、16S擴(kuò)增子序列肴捉、蛋白序列比對(duì)基因注釋腹侣、物種注釋 、基因功能注釋數(shù)據(jù)庫(kù)齿穗。Blast準(zhǔn)確性高傲隶，速度慢。Diamond速度更快窃页，常用于二代測(cè)序短序列比對(duì)跺株。
一、Diamond blastp
Diamond blastx功能可用于DNA序列比對(duì)【蛋白庫(kù)】
【核酸】比對(duì)【蛋白庫(kù)】
diamond blastx \
--db /path/to/protein.dmnd \
--query /path/to/genome.fna \
-e 1e-5 --outfmt 6 --more-sensitive --max-target-seqs 1 --threads 52 --quiet --id 80 --subject-cover 50 --query-cover 50 \
--out /path/to/anno.txt
【蛋白】比對(duì)【蛋白庫(kù)】
- diamond格式化蛋白庫(kù)
diamond makedb -p 52 \
--in /path/to/protein.fasta \
--db /path/to/protein.dmnd
- query比對(duì)格式化蛋白庫(kù)
- paraments
blastp 比蛋白
blastx 比核酸
blastp Align amino acid query sequences against a protein reference database
blastx Align DNA query sequences against a protein reference database
--query-cover minimum query cover% to report an alignment
--subject-cover minimum subject cover% to report an alignment
--out (-o) 輸出
--query (-q) 輸入
--db (-d) 格式化數(shù)據(jù)庫(kù)
--in Fasta數(shù)據(jù)庫(kù)
--quiet 安靜模式
--verbose (-v) 啰嗦模式
--outfmt (-f) 輸出格式
5 = BLAST XML
6 = BLAST tabular
100 = DIAMOND alignment archive (DAA)
101 = SAM
--top / --max-target-seqs 保留個(gè)數(shù)
--evalue (-e) 最大evalue
--id 最小一致性
--sensitive 敏感模式 (default: fast)
--more-sensitive 更敏感模式 (default: fast)
--block-size (-b) sequence block size in billions of letters (default=2.0)
--index-chunks (-c) number of chunks for index processing
- command
diamond blastp \
--db /path/to/protein.dmnd \
--query /path/to/genome.faa \
-e 1e-5 --outfmt 6 --more-sensitive --max-target-seqs 1 --threads 52 --quiet --id 60 \
--out /path/to/anno.txt
Diamond比對(duì)結(jié)果
1. qseqid | query sequence id
2. sseqid | subject sequence id
3. pident | percentage of identical matches
4. length | alignment length
5. mismatch | number of mismatches
6. gapopen | number of gap openings
7. q start | start of alignment in query
8. q end | end of alignment in query
9. s start | start of alignment in subject
10. s end | end of alignment in subject
11. evalue
12. bitscore
二腮出、Blastn & Blastp
Blastn【基因】比對(duì)【基因庫(kù)】
注釋信息可以是物種帖鸦，也可以是功能，比對(duì)就O了
- blast格式化數(shù)據(jù)庫(kù)
makeblastdb -in database.fna \
-input_type fasta -dbtype nucl -title prefix -parse_seqids \
-out /path/to/prefix
-dbtype <String, nucl, prot>
-input_type <String, asn1_bin, asn1_txt, blastdb, fasta>
-title <String> Title for BLAST database
-parse_seqids Option to parse seqid for FASTA input
-out <String> Name of BLAST database to be created
- query比對(duì)格式化數(shù)據(jù)庫(kù)
blastn -db /path/to/prefix \
-query /path/to/query.fasta \
-outfmt 6 \
-out /path/to/qcov80_id80_blast.out \
-num_threads 64 -perc_identity 80 -qcov_hsp_perc 80
-max_target_seqs: Maximum number of aligned sequences to keep. Default = 500
-evalue: E值就是Score值可靠性的評(píng)價(jià)胚嘲。
它表明在隨機(jī)的情況下作儿，其它序列與目標(biāo)序列相似度要大于S值的可能性。所以它的分值越低越好馋劈。
-perc_identity: 序列一致性
-qcov_hsp_perc <Real, 0..100> Percent query coverage per hsp草娜。HSP是high scoring pair
Blastp【蛋白】比對(duì)【蛋白庫(kù)】
- blast格式化數(shù)據(jù)庫(kù)
makeblastdb -in /path/to/pro.fas \
-input_type fasta -dbtype prot
-title blastn_pro \
-out /path/to/blastn_pro -parse_seqids
- query比對(duì)格式化數(shù)據(jù)庫(kù)
blastp -db /path/to/blastn_pro \
-query query.faa \
-out blast_pro.out \
-num_threads 52 -qcov_hsp_perc 80 -outfmt 6 \
-evalue 1e-5 -num_alignments 1
Blast比對(duì)結(jié)果
1、Query id：查詢序列ID標(biāo)識(shí)
2拐揭、Subject id：比對(duì)上的目標(biāo)序列ID標(biāo)識(shí)
3玄窝、% identity：序列比對(duì)的一致性百分比
4、alignment length：符合比對(duì)的比對(duì)區(qū)域的長(zhǎng)度
5械姻、mismatches：比對(duì)區(qū)域的錯(cuò)配數(shù)
6妒蛇、gap openings：比對(duì)區(qū)域的gap數(shù)目
7、q start：Query id起始位點(diǎn)
8、q end：Query id終止位點(diǎn)
9绣夺、s start：Subject id起始位點(diǎn)
10吏奸、s end：Subject id終止位點(diǎn)
11、e value：比對(duì)結(jié)果的期望值
12陶耍、bit score：比對(duì)結(jié)果的bit score值
https://github.com/bbuchfink/diamond/wiki
The sensitivity can be adjusted using the options:
--mid-sensitive,
--sensitive,
--more-sensitive,
--very-sensitive,
--ultra-sensitive.
Diamond 比對(duì)軟件使用
The default e-value cutoff of DIAMOND is 0.001 while that of BLAST is 10, so by default the program will search a lot more stringently than BLAST and not report weak hits.
三奋蔚、比對(duì)軟件默認(rèn)取值
prokka
https://github.com/tseemann/prokka/blob/master/README.md
--evalue [n.n] Similarity e-value cut-off (default '1e-06')
default '1e-06'
diamond
default e-value cutoff of DIAMOND is 0.001 while that of BLAST is 10
1e-3
blast
default e-value cutoff of DIAMOND is 0.001 while that of BLAST is 10
10
kofamscan
-E, --e-value <e_value> Largest E-value required of the hits
親自測(cè)試是 1e-3
????
