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云之南

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专业背景:计算机科学 研究方向与兴趣: JavaEE-Web软件开发, 生物信息学, 数据挖掘与机器学习, 智能信息系统 目前工作: 基因组, 转录组, NGS高通量数据分析, 生物数据挖掘, 植物系统发育和比较进化基因组学

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BLAST+实战  

2010-07-19 16:00:31|  分类: 生物信息学 |  标签: |举报 |字号 订阅

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BLAST+实战
2010-04-29 16:08
http://hi.baidu.com/lidaof/blog/item/9d29eade7f340a5895ee3724.html

项目要求,开始使用BLAST+

我们从格式化数据库开始

1,我先获得了一个植物rRNA的序列文件,为fasta格式,用makeblastdb格式化之,如下

makeblastdb -in plant26.rna.fna -dbtype nucl -parse_seqids  -hash_index  -out plant_rna

注意:在BLAST+2.2.24中 不加参数 -parse_seqids ,-hash_index  不然成死循环


Building a new DB, current time: 04/29/2010 16:09:11
New DB name:   plant_rna
New DB title: plant26.rna.fna
Sequence type: Nucleotide
Keep Linkouts: T
Keep MBits: T
Maximum file size: 1073741824B
Adding sequences from FASTA; added 6747 sequences in 0.546889 seconds.

此时目录下还是如往常一下生成如下文件

plant_rna.nhr plant_rna.nin plant_rna.nnd plant_rna.nni plant_rna.nsd plant_rna.nsi plant_rna.nsq

2,我们有如下一个fasta序列想做blast,文件如下

more test
>gi|145342129|ref|XM_001416109.1| Ostreococcus lucimarinus CCE9901 vacuolar ATP synthase subunit D, probab
le (VaoD) mRNA, complete cds
ATGTTCAACGCGAAGAACGGTTTTTCTGAGGCACACGTGAGGGGATGTCAGACCAAACGACTCACCAAACAGAACTACGC
CGAACTTTCTCGATGTGACACGTTGGAAGACATCAAGACGTACTTGCAAACGATGAGTGATTATTCAGAATATGTTCGTG
ATCTTCAAGCGCCAGTGAGACCGGTTGACATTATTGAATGCTGCAGAAAGAGACAGATCGCAGAGTTTAATATTTGCTGT
CAGCAGGCTTCTTCCCCTTTGTCCAATTTTTTGGAGTATTTGACGTACGGATACATGATCGATAATCTTGTGTTGGCTTT
AAATGGCATGCTTCGTGGACGTACCACAGAGGCAATACTTGAGAAGTGTAGCCCCATTGGTTTTTTCGATTCTTTATCCG
CGGTTGTCGTGTCGAGTAGTGTCCAAGAACTCTACAGACTAGCTCTCGTGGATACACCGCTTGCCTCTTATTTCAGTAGC
TCGATTAAGGCAGAAGATCTGGATGAGTTAAATATTGAGCTCATACGGAACGTCCTATACAAGGAATATTTGCAAGATTT
CATGGTTTTCTGCAACAAAATGGATCAAAACACACGTCAATTGATGGAGAAACTACTTAGCATGGAGGCCGATCGGCACG
CGATAAGAATCACACTGAACTCTTTCGGAACAGAGCTTTCCAAGGCTGATCGAAGAAATCTTTATACGAATTTTGGCACC
ATGTACCCCGATGGCTTCGCGCGTCTTGCGAATTGTGAAACGGTAGATGAAGTGAAACGCATACTAGTAGCTTATCCAGA
ATTCAGAGAGTTGACGAAAAGTGATGATCCCCACTACATTGACAGGGGACTACGCGTTCTCGAACTGGAAGCATGTGGAC
AAGCACTCGATGAGCAATTCAATTTCGCTATCTTTTATGCTTTCGTAAAGTTTCAGGAGAACGAAATAAACAACCTGATG
TGGCTCACTGAGTGTGTTGCTCAAAGGCAAAAAAGTAGTCTAGGCGAGGGCATTGTCTACATACAATAG

3,blast搜索命令格式如下:

blastn -db plant_rna -query test -out test.blastn

4,我们来看看输入文件test.blastn的内容

BLASTN 2.2.23+


Reference: Zheng Zhang, Scott Schwartz, Lukas Wagner, and Webb
Miller (2000), "A greedy algorithm for aligning DNA sequences", J
Comput Biol 2000; 7(1-2):203-14.

Database: plant26.rna.fna
           6,747 sequences; 8,392,753 total letters

Query= gi|145342129|ref|XM_001416109.1| Ostreococcus lucimarinus CCE9901
vacuolar ATP synthase subunit D, probale (VaoD) mRNA, complete cds
Length=1029
                                                                      Score     E
Sequences producing significant alignments:                          (Bits) Value

ref|XM_001416109.1| Ostreococcus lucimarinus CCE9901 vacuolar AT... 1901    0.0


>ref|XM_001416109.1| Ostreococcus lucimarinus CCE9901 vacuolar ATP synthase subunit
D, probable (VaoD) mRNA, complete cds
Length=1029

Score = 1901 bits (1029), Expect = 0.0
Identities = 1029/1029 (100%), Gaps = 0/1029 (0%)
Strand=Plus/Plus

Query 1     ATGTTCAACGCGAAGAACGGTTTTTCTGAGGCACACGTGAGGGGATGTCAGACCAAACGA 60
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1     ATGTTCAACGCGAAGAACGGTTTTTCTGAGGCACACGTGAGGGGATGTCAGACCAAACGA 60

Query 61    CTCACCAAACAGAACTACGCCGAACTTTCTCGATGTGACACGTTGGAAGACATCAAGACG 120
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 61    CTCACCAAACAGAACTACGCCGAACTTTCTCGATGTGACACGTTGGAAGACATCAAGACG 120

Query 121   TACTTGCAAACGATGAGTGATTATTCAGAATATGTTCGTGATCTTCAAGCGCCAGTGAGA 180
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 121   TACTTGCAAACGATGAGTGATTATTCAGAATATGTTCGTGATCTTCAAGCGCCAGTGAGA 180

Query 181   CCGGTTGACATTATTGAATGCTGCAGAAAGAGACAGATCGCAGAGTTTAATATTTGCTGT 240
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 181   CCGGTTGACATTATTGAATGCTGCAGAAAGAGACAGATCGCAGAGTTTAATATTTGCTGT 240

Query 241   CAGCAGGCTTCTTCCCCTTTGTCCAATTTTTTGGAGTATTTGACGTACGGATACATGATC 300
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 241   CAGCAGGCTTCTTCCCCTTTGTCCAATTTTTTGGAGTATTTGACGTACGGATACATGATC 300

Query 301   GATAATCTTGTGTTGGCTTTAAATGGCATGCTTCGTGGACGTACCACAGAGGCAATACTT 360
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 301   GATAATCTTGTGTTGGCTTTAAATGGCATGCTTCGTGGACGTACCACAGAGGCAATACTT 360

Query 361   GAGAAGTGTAGCCCCATTGGTTTTTTCGATTCTTTATCCGCGGTTGTCGTGTCGAGTAGT 420
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 361   GAGAAGTGTAGCCCCATTGGTTTTTTCGATTCTTTATCCGCGGTTGTCGTGTCGAGTAGT 420

Query 421   GTCCAAGAACTCTACAGACTAGCTCTCGTGGATACACCGCTTGCCTCTTATTTCAGTAGC 480
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 421   GTCCAAGAACTCTACAGACTAGCTCTCGTGGATACACCGCTTGCCTCTTATTTCAGTAGC 480

Query 481   TCGATTAAGGCAGAAGATCTGGATGAGTTAAATATTGAGCTCATACGGAACGTCCTATAC 540
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 481   TCGATTAAGGCAGAAGATCTGGATGAGTTAAATATTGAGCTCATACGGAACGTCCTATAC 540

Query 541   AAGGAATATTTGCAAGATTTCATGGTTTTCTGCAACAAAATGGATCAAAACACACGTCAA 600
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 541   AAGGAATATTTGCAAGATTTCATGGTTTTCTGCAACAAAATGGATCAAAACACACGTCAA 600

Query 601   TTGATGGAGAAACTACTTAGCATGGAGGCCGATCGGCACGCGATAAGAATCACACTGAAC 660
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 601   TTGATGGAGAAACTACTTAGCATGGAGGCCGATCGGCACGCGATAAGAATCACACTGAAC 660

Query 661   TCTTTCGGAACAGAGCTTTCCAAGGCTGATCGAAGAAATCTTTATACGAATTTTGGCACC 720
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 661   TCTTTCGGAACAGAGCTTTCCAAGGCTGATCGAAGAAATCTTTATACGAATTTTGGCACC 720

Query 721   ATGTACCCCGATGGCTTCGCGCGTCTTGCGAATTGTGAAACGGTAGATGAAGTGAAACGC 780
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 721   ATGTACCCCGATGGCTTCGCGCGTCTTGCGAATTGTGAAACGGTAGATGAAGTGAAACGC 780

Query 781   ATACTAGTAGCTTATCCAGAATTCAGAGAGTTGACGAAAAGTGATGATCCCCACTACATT 840
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 781   ATACTAGTAGCTTATCCAGAATTCAGAGAGTTGACGAAAAGTGATGATCCCCACTACATT 840

Query 841   GACAGGGGACTACGCGTTCTCGAACTGGAAGCATGTGGACAAGCACTCGATGAGCAATTC 900
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 841   GACAGGGGACTACGCGTTCTCGAACTGGAAGCATGTGGACAAGCACTCGATGAGCAATTC 900

Query 901   AATTTCGCTATCTTTTATGCTTTCGTAAAGTTTCAGGAGAACGAAATAAACAACCTGATG 960
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 901   AATTTCGCTATCTTTTATGCTTTCGTAAAGTTTCAGGAGAACGAAATAAACAACCTGATG 960

Query 961   TGGCTCACTGAGTGTGTTGCTCAAAGGCAAAAAAGTAGTCTAGGCGAGGGCATTGTCTAC 1020
             ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 961   TGGCTCACTGAGTGTGTTGCTCAAAGGCAAAAAAGTAGTCTAGGCGAGGGCATTGTCTAC 1020

Query 1021 ATACAATAG 1029
             |||||||||
Sbjct 1021 ATACAATAG 1029

Lambda     K      H
    1.33    0.621     1.12

Gapped
Lambda     K      H
    1.28    0.460    0.850

Effective search space used: 8286997432


Database: plant26.rna.fna
    Posted date: Apr 29, 2010 4:09 PM
Number of letters in database: 8,392,753
Number of sequences in database: 6,747

Matrix: blastn matrix 1 -2
Gap Penalties: Existence: 0, Extension: 0

5,我们可以看出,如果不加任何参数,和以前的输出差不多相同,大家还是可以使用以前写的bioperl或biopython parser进行结果的分析

6,下面我们看看增加输出参数呢,我们使用-outfmt 7作为控制输出结果,其中选择性输出部分内容,命令和输出如下:

blastn -db plant_rna -query test -outfmt "7 qacc sacc evalue length pident"
# BLASTN 2.2.23+
# Query: gi|145342129|ref|XM_001416109.1| Ostreococcus lucimarinus CCE9901 vacuolar ATP synthase subunit D, probale (VaoD) mRNA, complete cds
# Database: plant_rna
# Fields: query acc., subject acc., evalue, alignment length, % identity
# 1 hits found
gi|145342129|ref|XM_001416109.1|        XM_001416109    0.0     1029    100.00
# BLAST processed 1 queries

这个文件是自解释的,大家可以看得很清楚每项是什么意思,呵呵,其他输出的一些部分特性如下:

blastn -db plant_rna -query test -outfmt "7 qacc sacc evalue length pident"
# BLASTN 2.2.23+
# Query: gi|145342129|ref|XM_001416109.1| Ostreococcus lucimarinus CCE9901 vacuolar ATP synthase subunit D, probale (VaoD) mRNA, complete cds
# Database: plant_rna
# Fields: query acc., subject acc., evalue, alignment length, % identity
# 1 hits found
gi|145342129|ref|XM_001416109.1|        XM_001416109    0.0     1029    100.00
# BLAST processed 1 queries

blastn -db plant_rna -query test -outfmt "7 qgi sgi evalue length pident"   # BLASTN 2.2.23+
# Query: gi|145342129|ref|XM_001416109.1| Ostreococcus lucimarinus CCE9901 vacuolar ATP synthase subunit D, probale (VaoD) mRNA, complete cds
# Database: plant_rna
# Fields: query gi, subject gi, evalue, alignment length, % identity
# 1 hits found
0       145342129       0.0     1029    100.00
# BLAST processed 1 queries

blastn -db plant_rna -query test -outfmt "7 qid sid evalue length pident"
# BLASTN 2.2.23+
# Query: gi|145342129|ref|XM_001416109.1| Ostreococcus lucimarinus CCE9901 vacuolar ATP synthase subunit D, probale (VaoD) mRNA, complete cds
# Database: plant_rna
# Fields: evalue, alignment length, % identity
# 1 hits found
0.0     1029    100.00
# BLAST processed 1 queries

blastn -db plant_rna -query test -outfmt "7 qseqid sseqid evalue length pident"
# BLASTN 2.2.23+
# Query: gi|145342129|ref|XM_001416109.1| Ostreococcus lucimarinus CCE9901 vacuolar ATP synthase subunit D, probale (VaoD) mRNA, complete cds
# Database: plant_rna
# Fields: query id, subject id, evalue, alignment length, % identity
# 1 hits found
gi|145342129|ref|XM_001416109.1|        gi|145342129|ref|XM_001416109.1|        0.0     1029    100.00
# BLAST processed 1 queries

blastn -db plant_rna -query test -outfmt "7 qframe sseqid evalue length pident"
# BLASTN 2.2.23+
# Query: gi|145342129|ref|XM_001416109.1| Ostreococcus lucimarinus CCE9901 vacuolar ATP synthase subunit D, probale (VaoD) mRNA, complete cds
# Database: plant_rna
# Fields: query frame, subject id, evalue, alignment length, % identity
# 1 hits found
1       gi|145342129|ref|XM_001416109.1|        0.0     1029    100.00


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