blast | diamond 输出结果选择和解析 | 比对

之前的文章：构建NCBI本地BLAST数据库 (NR NT等) | blastx/diamond使用方法 | blast构建索引 | makeblastdb

本地运行blast时，需要指定out format。

常见的网页版blast结果可以参照：Blast结果的详细解析

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*** Formatting options  -outfmt <String>    alignment view options:      0 = Pairwise,      1 = Query-anchored showing identities,      2 = Query-anchored no identities,      3 = Flat query-anchored showing identities,      4 = Flat query-anchored no identities,      5 = BLAST XML,      6 = Tabular,      7 = Tabular with comment lines,      8 = Seqalign (Text ASN.1),      9 = Seqalign (Binary ASN.1),     10 = Comma-separated values,     11 = BLAST archive (ASN.1),     12 = Seqalign (JSON),     13 = Multiple-file BLAST JSON,     14 = Multiple-file BLAST XML2,     15 = Single-file BLAST JSON,     16 = Single-file BLAST XML2,     18 = Organism Report      Options 6, 7 and 10 can be additionally configured to produce    a custom format specified by space delimited format specifiers.    The supported format specifiers are:         qseqid means Query Seq-id            qgi means Query GI           qacc means Query accesion        qaccver means Query accesion.version           qlen means Query sequence length         sseqid means Subject Seq-id      sallseqid means All subject Seq-id(s), separated by a ';'            sgi means Subject GI         sallgi means All subject GIs           sacc means Subject accession        saccver means Subject accession.version        sallacc means All subject accessions           slen means Subject sequence length         qstart means Start of alignment in query           qend means End of alignment in query         sstart means Start of alignment in subject           send means End of alignment in subject           qseq means Aligned part of query sequence           sseq means Aligned part of subject sequence         evalue means Expect value       bitscore means Bit score          score means Raw score         length means Alignment length         pident means Percentage of identical matches         nident means Number of identical matches       mismatch means Number of mismatches       positive means Number of positive-scoring matches        gapopen means Number of gap openings           gaps means Total number of gaps           ppos means Percentage of positive-scoring matches         frames means Query and subject frames separated by a '/'         qframe means Query frame         sframe means Subject frame           btop means Blast traceback operations (BTOP)         staxid means Subject Taxonomy ID       ssciname means Subject Scientific Name       scomname means Subject Common Name     sblastname means Subject Blast Name      sskingdom means Subject Super Kingdom        staxids means unique Subject Taxonomy ID(s), separated by a ';'              (in numerical order)      sscinames means unique Subject Scientific Name(s), separated by a ';'      scomnames means unique Subject Common Name(s), separated by a ';'     sblastnames means unique Subject Blast Name(s), separated by a ';'              (in alphabetical order)     sskingdoms means unique Subject Super Kingdom(s), separated by a ';'              (in alphabetical order)         stitle means Subject Title     salltitles means All Subject Title(s), separated by a '<>'        sstrand means Subject Strand          qcovs means Query Coverage Per Subject        qcovhsp means Query Coverage Per HSP         qcovus means Query Coverage Per Unique Subject (blastn only)    When not provided, the default value is:    'qaccver saccver pident length mismatch gapopen qstart qend sstart send    evalue bitscore', which is equivalent to the keyword 'std'    Default = `0'

默认是0，也就是会输出比对的结果。

但是这样的结果显然不适合批量处理，批量处理的文件格式显然必须是dataframe。

所以网上有人推荐“outfmt 7 or 10 works perfect”，所以一般就选10吧。

Diamond的输出格式：

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--outfmt (-f)          output format     0   = BLAST pairwise     5   = BLAST XML     6   = BLAST tabular     100 = DIAMOND alignment archive (DAA)     101 = SAM       Value 6 may be followed by a space-separated list of these keywords:       qseqid means Query Seq - id     qlen means Query sequence length     sseqid means Subject Seq - id     sallseqid means All subject Seq - id(s), separated by a ';'     slen means Subject sequence length     qstart means Start of alignment in query     qend means End of alignment in query     sstart means Start of alignment in subject     send means End of alignment in subject     qseq means Aligned part of query sequence     sseq means Aligned part of subject sequence     evalue means Expect value     bitscore means Bit score     score means Raw score     length means Alignment length     pident means Percentage of identical matches     nident means Number of identical matches     mismatch means Number of mismatches     positive means Number of positive - scoring matches     gapopen means Number of gap openings     gaps means Total number of gaps     ppos means Percentage of positive - scoring matches     qframe means Query frame     btop means Blast traceback operations(BTOP)     staxids means unique Subject Taxonomy ID(s), separated by a ';' (in numerical order)     stitle means Subject Title     salltitles means All Subject Title(s), separated by a '<>'     qcovhsp means Query Coverage Per HSP     qtitle means Query title       Default: qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore

diamond就选6吧，便于批量处理。

 

diamond 比对转录本到Pfam库的部分结果，可以看到，格式6非常适合做批量处理。

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wgs.RNAseq_00000687     M1AFS7.1        60.9    179     65      2       337     861     102     279     8.1e-55 221.5 wgs.RNAseq_00000687     A0A0B2PIQ0.1    61.0    177     66      2       337     861     319     494     4.0e-54 219.2 wgs.RNAseq_00000687     A0A0L9UG27.1    61.2    178     65      3       337     861     315     491     4.0e-54 219.2 wgs.RNAseq_00000687     A0A0L9UG27.1    63.2    38      14      0       6       119     250     287     1.0e-04 55.1 wgs.RNAseq_00000688     A0A0D2QMP5.1    65.8    114     35      2       218     550     317     429     1.7e-34 153.3 wgs.RNAseq_00000688     A0A0D2QMP5.1    53.2    62      26      1       36      221     256     314     8.7e-10 71.2 wgs.RNAseq_00000688     A0A0D2TR34.1    65.8    114     35      2       218     550     322     434     1.7e-34 153.3 wgs.RNAseq_00000688     A0A0D2TR34.1    53.2    62      26      1       36      221     261     319     8.7e-10 71.2 wgs.RNAseq_00000688     F6H0G0.1        65.8    111     36      2       221     550     320     429     5.6e-33 148.3

 

每一列是什么呢？ BLASTn output format 6

Column headers:
qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore

 

1.	qseqid	query (e.g., gene) sequence id
2.	sseqid	subject (e.g., reference genome) sequence id
3.	pident	percentage of identical matches
4.	length	alignment length
5.	mismatch	number of mismatches
6.	gapopen	number of gap openings
7.	qstart	start of alignment in query
8.	qend	end of alignment in query
9.	sstart	start of alignment in subject
10.	send	end of alignment in subject
11.	evalue	expect value
12.	bitscore	bit score

 

 

比对结果的第三列和第四列非常有用，尤其是在鉴别软件stringtie等预测出来的转录本是否为有效转录本时，其实预测出来的转录本大部分都是没有意义的，但是又能部分hit到蛋白上，这时我们就只能选出比对最长的那个转录本，其余的可以看作是无效的转录本。

操作比较简单，先对比对长度排个序，从长到短。

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1	cat extract_no_N_200.fasta.diamond.nr | sort -n -r -k4 > extract_no_N_200.fasta.diamond.nr.sort

其次就是利用python的panda模块去冗余就好了。

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import pandas as pd   infile = "extract_no_N_200.fasta.diamond.nr.sort" #infile = "test.sort"   df = pd.read_csv(infile, sep="\t", header=None) df.columns = ["l1","l2","l3","l4","l5","l6","l7","l8","l9","l10","l11","l12"]   df1 = df.sort_values(['l4'],ascending=False)   df2 = df1.drop_duplicates(subset=[df1.columns[1]], keep = 'first')   df2.to_csv("rm_dup_protein_"+infile, sep="\t", index=False, header=False)   df3 = df2.sort_values(['l3'],ascending=False)   df4 = df3.drop_duplicates(subset=[df3.columns[0]], keep = 'first')   df4.to_csv("unique_protein_"+infile, sep="\t", index=False, header=False)

　　

这样我们得到的转录本就基本上是无冗余的了。

 

然后对每个转录本取identical match比例最高的蛋白就好了。其实信息是可以全部保留的。

blast和diamond怎么只输出最优的hit？

 

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1	blastn -query transcripts.fa -out transcripts.blast.txt -task megablast -db refseq_rna -num_threads 12 -evalue 1e-10 -best_hit_score_edge 0.05 -best_hit_overhang 0.25 -outfmt 7 -perc_identity 50 -max_target_seqs 1

　　

 

待续~