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Old 01-11-2011, 06:32 AM   #1
anna_
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Default extract data from fasta-files with perl??

Hello,

I'm very knew to the subject and this is my first question ever:

I'm working with 454 data. I translated my contigs to protein sequences, created a blastdatabase with theses and used a bunch of downloaded proteins as a query.

Now I have an output file with "hit-contigs". My aim is to extract theses hit-contigs from the original fasta-file with all contigs.

As far as I'm informed things like that can be done automatically by applying perl scripts. Unfortuneately I have no idea how.

I'm very thankful for help an of course open to learn

anna
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Old 01-12-2011, 07:59 AM   #2
simonandrews
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I guess the general strategy would be:
  1. Read all of your hit contig names into a hash
  2. Read through your fasta sequences one at a time
  3. If the contig name matches one of your hit contigs then keep it, otherwise throw it away

If you have the BioPerl modules installed then reading through a fasta file and writing out selected entries is pretty trivial.
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Old 01-12-2011, 11:26 AM   #3
kmcarr
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Quote:
Originally Posted by anna_ View Post
Hello,

I'm very knew to the subject and this is my first question ever:

I'm working with 454 data. I translated my contigs to protein sequences, created a blastdatabase with theses and used a bunch of downloaded proteins as a query.

Now I have an output file with "hit-contigs". My aim is to extract theses hit-contigs from the original fasta-file with all contigs.

As far as I'm informed things like that can be done automatically by applying perl scripts. Unfortuneately I have no idea how.

I'm very thankful for help an of course open to learn

anna
Anna,

You can do this yourself and it would be a good learning exercise, but since you have already made a BLAST database of the contigs, NCBI has kindly provided tools for doing exactly what you want.

Create a text file of the contig IDs you want to extract, one ID per line, no other information in the file. Be careful to use the same IDs as BLAST for your contigs. We'll call this file "myContigList.txt".

The command to use depends on whether you are using the old school (C-Toolkit) BLAST or the new BLAST+. These ancillary commands should have been installed when you installed BLAST

Old school use the command "fastacmd"

Code:
$ fastacmd -d myBlastDBName -p protein -i myContigList.txt -o myHitContigs.fasta
You can omit the '-p protein' and let the command guess the DB type.

For the new BLAST+ distribution use "blastdbcmd"

Code:
$ blastdbcmd -db myBlastDBName -dbtype prot -entry_batch myContigList.txt -outfmt %f -out myHitContigs.fasta
Again you could omit the '-dbtype prot' and let the program guess. The -outfmt %f tells the program to output sequences in FASTA format; you could also omit this since this is the default output format.
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Old 01-13-2011, 06:56 AM   #4
anna_
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Thank you both,

I'm trying and the NBCI tool solution:

I use blast+. To get the text-file with the ID I used the command -outfmt "10 qgi". I when I use this output as the entry batch file I get the massage: "no valid entries to search". I added a ">" to every ID, but still there is the same error.

I don't know what's wrong.
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Old 01-14-2011, 01:08 PM   #5
kmcarr
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Quote:
Originally Posted by anna_ View Post
Thank you both,

I'm trying and the NBCI tool solution:

I use blast+. To get the text-file with the ID I used the command -outfmt "10 qgi". I when I use this output as the entry batch file I get the massage: "no valid entries to search". I added a ">" to every ID, but still there is the same error.

I don't know what's wrong.
Anna,

You don't want to use outfmt 10 (comma separated values), you want outfmt 6 (tabular data w/o headers). Also you don't want to output the query gi (qgi), you want to report the accessions of the hits (subject) you find. (Your hits won't have gi's since you are working with a custom database, not an NCBI db.) You also want to capture all of the hits for your query so sallacc is the outfmt you want.

Your blastx command should look like

Code:
$ blastx -db myDB -query myQuery -out myContigList.txt -outfmt "6 sallacc" [other blastx options]
If you had multiple sequences in your query file the resultant myContigList.txt file may have the same contig ID listed more than once (more than one of your queries may have match the same contig). To eliminate the redundancies in this list you can use the sort command with the unique filter.

Code:
$ sort -u myContigList.txt > myContigList_uniq.txt
You could now use the myContigList_uniq.txt file in the blastdbcmd described in my earlier post.

Hope this helps.
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Old 02-17-2011, 12:28 AM   #6
anna_
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Thank you,

it works. I feel quite a bit ashamed because of my stupidity but I'll go on with asking and learning (hopefully), because I have no other choice.

thanks again!
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Old 02-17-2011, 02:29 AM   #7
aliealexandre
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Hi everybody,

Thank you, Anna, I wanted to ask exactly the same question !

Thank you, kmcarr, for you detailed answer. But even I rigourously followed your wy, I get the "no valide entries to search" message....

What could be the reason ? Do you have any idea ?

Thank you for your help

Alex
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Old 02-17-2011, 05:25 AM   #8
kmcarr
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Quote:
Originally Posted by aliealexandre View Post
Hi everybody,

Thank you, Anna, I wanted to ask exactly the same question !

Thank you, kmcarr, for you detailed answer. But even I rigourously followed your wy, I get the "no valide entries to search" message....

What could be the reason ? Do you have any idea ?

Thank you for your help

Alex
Alex,

Can you post a few lines of the list file containing the accessions you are trying to look up. Also post a few lines from the following command:

Code:
blastdbcmd -db <your_blast_db_name> -outfmt %a -entry all
Does the format of the accessions in your query file look like the accessions as they appear in the BLAST database?
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Old 02-17-2011, 05:51 AM   #9
jordi
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If I use formatdb command instead makeblastdb, I should use the -o T option, right? I didn't know why my sequences were'nt found in a certain database...
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Old 02-17-2011, 06:08 AM   #10
kmcarr
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Quote:
Originally Posted by jordi View Post
If I use formatdb command instead makeblastdb, I should use the -o T option, right? I didn't know why my sequences were'nt found in a certain database...
Yes, I believe that is true. Without using "-o T" there will be no index of the SeqIDs (accessions) for fastacmd to look up accessions in. You would have to use gi numbers (if they existed in the original fasta file) to retrieve sequences.
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Old 02-17-2011, 06:51 PM   #11
aliealexandre
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kmcarr

These are some lines from the blastdbcmd command :

BL_ORD_ID:0
BL_ORD_ID:1
BL_ORD_ID:2
BL_ORD_ID:3
BL_ORD_ID:4
BL_ORD_ID:5

And these from the text file comtaining accessions :

jgi|Nemve1|24|gw.52.2.1
jgi|Nemve1|45|gw.188.2.1
jgi|Nemve1|45|gw.188.2.1
jgi|Nemve1|45|gw.188.2.1
jgi|Nemve1|45|gw.188.2.1
jgi|Nemve1|45|gw.188.2.1

Of course these ID are exactly the same than in my db

Thank you very much for your help

Alex
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Old 02-18-2011, 05:43 AM   #12
kmcarr
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Quote:
Originally Posted by aliealexandre View Post
kmcarr

These are some lines from the blastdbcmd command :

BL_ORD_ID:0
BL_ORD_ID:1
BL_ORD_ID:2
BL_ORD_ID:3
BL_ORD_ID:4
BL_ORD_ID:5

And these from the text file comtaining accessions :

jgi|Nemve1|24|gw.52.2.1
jgi|Nemve1|45|gw.188.2.1
jgi|Nemve1|45|gw.188.2.1
jgi|Nemve1|45|gw.188.2.1
jgi|Nemve1|45|gw.188.2.1
jgi|Nemve1|45|gw.188.2.1

Of course these ID are exactly the same than in my db

Thank you very much for your help

Alex
Alex,

The format of the accessions in your query file (jgi|Nemve1|45|gw.188.2.1) differs from the accessions in your BLASTdb (BL_ORD_ID:5) which is why the search is failing.

Fortunately jordi's question above reminded me of an important point which explains why. When a BLASTdb is created the default behavior is to NOT parse and index the accessions (Seq-IDs in BLAST terminology). In that case generic seq_ids will be created by the program (e.g. BL_ORD_ID:0). Without this index of accessions you can't perform searches by accession. You need to make a new BLASTdb but add the option "-parse_seqids" to your makeblastdb command.

I noticed that you list the same accession 5 times in your query list (jgi|Nemve1|45|gw.188.2.1), why is that? See my post above about how to make your the entries in your query list unique using the sort command. Also, while it is acceptable to use the full id string (jgi|Nemve1|45|gw.188.2.1) for your query, you can use just the accession portion of the name, gw.188.2.1, to achieve the same result.
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Old 02-20-2011, 09:22 PM   #13
aliealexandre
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Great !! it works

Thank you, kmcarr, for your very helpfull and very understandable advices.

See you

Alex
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Old 02-23-2011, 01:08 AM   #14
anna_
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kmcarr, another question about makeblastdb from anna again this time:

I should explain the procedure at first:

I'm searching for proteins that are related to drought stress in a huge conifer transcriptome.

I searched the UniProt and the NCBI databases for proteins with "drought" as a keyword and downloaded the hits in as fasta-files. I concatenated the two files with the unix cat command. I guess a lot of Proteins will appear many fold in the concatenated file and that will have consequences for my blast statistics. So I applied a perl script to remove redundancy. Since I cannot write perl myself (I'm learning), I took the script from the O'reilly BLAST book.

#!/usr/bin/perl
use Bio::SeqIO;

my %NR;
my $file = Bio::SeqIO->new(-fh => \*ARGV);
while (my $fasta = $file->next_seq){
my $def = $fasta->id ." ".$fasta->desc;
$NR{$fasta->seq}{$def} = 1;
}
for my $seq(keys %NR){
print ">", join(chr(1),keys
%{$NR{$seq}}),"\n",$seq,"\n";
}

After that the sequences were unique and the identifiers were all in one line after one another:

>gi|57908279|gb|AAW59263.1| water-stress inducible protein 3 [Pinus taeda]gi|57908277|gb|AAW59262.1| water-stress inducible protein 3 [Pinus taeda]gi|57908297|gb|AAW59272.1| water-stress inducible protein 3 [Pinus taeda]gi|57908325|gb|AAW59286.1| water-stress inducible protein 3 [Pinus taeda]gi|57908331|gb|AAW59289.1| water-stress inducible protein 3 [Pinus taeda]
DENDNPPSEVVYSETTTAYGDEVIQSSDVYATGNVNSDEYEKARKEEKHHKHMEEVGGLGTMATGAFALHEKHAEKKDPEHAHRHKIEEEIAAAAAVGEGG

I was happy and applied:

makeblastdb -in protein_drought_db_uniq -dbtype prot -parse_seqids -logfile protein_drought_db_uniq.logfile > protein_drought_db_uniq

and then I got:

Blast database creation error: Error: Duplicate seq_ids are found.

Do you or anybody else have an idea how solve this dilemma?
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Old 02-23-2011, 02:32 AM   #15
jordi
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Very interesting question Anna!
I think it could be very useful to customize Uniprot data in order to obtain a column with the NCBI cross-reference ID, since Uniprot allows this option.. but I tried it and it doesn't work! I was unable to obtain the NCBI id's
Maybe, a Blast using one dataset as db and the other one as query could be useful parsing a 100% identity alignment with a given alignment length...
Or what about this??
http://www.uniprot.org/help/mapping
I mean, you should obtain a list as follows:
From To
Q9M2S6 AL132975
Q5BPS3 AC00716
FROM Uniprot Id's you got TO NCBi's Id's.

hope this helps.

Last edited by jordi; 02-23-2011 at 02:40 AM.
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Old 01-18-2012, 11:20 AM   #16
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Hi,
I am using blast+. I have formatted a nucleotide database using makeblastdb.

I am trying to extract sequences from a file containing a list of IDs.

Using the following:
Code:
/Users/wolniaklab/blast/programs/blastdbcmd -db /Users/wolniaklab/Desktop/search/seqs2 -dbtype nucl -entry_batch /Users/wolniaklab/Desktop/search/ids1.txt -out /Users/wolniaklab/Desktop/search/output.txt
When I do I get the same error for every ID I am searching for (here is an example):

Code:
Error: >lcl|comp9999_c1_seq11: OID not found
My list of ids is in this format:
Code:
>lcl|comp10021_c0_seq1
>lcl|comp1002_c0_seq1
>lcl|comp10045_c0_seq13
>lcl|comp10045_c0_seq14
>lcl|comp10045_c0_seq19
>lcl|comp10045_c0_seq4
>lcl|comp10045_c0_seq4
>lcl|comp10049_c0_seq4
>lcl|comp10075_c0_seq13
>lcl|comp10075_c0_seq9
>lcl|comp100777_c0_seq1
>lcl|comp10082_c0_seq1
The fast file I made the database from looks like this

Code:
>lcl|comp11191_c0_seq1 len=589 path=[0:0-128 613:129-135 136:136-588]
GTTCTATTGTATTGTTATCCATCTGAGGTTTTCTCTCTGCGTTTGTCTGTGCAGAATCTA
GTGATCTCCCACAACATGATGTGGCCACCAGGGATGGAACAAAGCTGGTGAGAAGGGCCG
ATATGGCTCGAAAAATTCCTCAATTCAAGATACTTTGATCCCTGCACCGAGCACCACTTC
AACAAAAATGAGAAAAACCATTTCTGCATTTGTTGTAATGAAGGTCCTCACTCCCATCAC
CAAACTCTCCAAGTCCGCCGGGCGTCCCATGCCAACTGTGTCCGGGTCGAAAACATCTCC
TAGATTCTAGACATTTCTGGAATTCAAACCTACATCATCAACAACCATAAAATTGTCTTC
CTCCAAAGGCAGGCCAATGTGAAGCAGATCATGTCAAGGTTGTTGATCAGTTCAACAGGA
GGTCTCCATGTCTCTGCTAATGCCAAGCATTGCCATACCTGTGGAAGAGCTTTGTCCACT
GATTTAATGAAGTTTTGCTCCATTAAATGCAAGCTTATGCCTACTTCTTTTAATTTTGTT
TCTAGAATTTGAAACTCATTTTACTAAACTGGTTATATTTTGTTTTTAG
>lcl|comp10877_c0_seq1 len=1212 path=[3176:0-121 3368:122-148 3395:149-192 3439:193-281 4481:282-332 3578:333-1211]
AAAGCATGCCTAAGTCGATTTATTATTAATTTATTTAGTCGCTTTATTCTAACTATCCCG
ACTCAAGCTTAACTAACGGTTCTACTATTCGATTTCCATCTCTAGGTTCGGTTTCTAACT
CGTCTAACTCCCTCGCCTACGGAATTCATGACTTCGGTCATCGCTAACCTCGGCAACCCT
CTACGTGAGTTTAGTCACCAACAGTGTCAAGTTCCGTCCAACAGCGTCAACATCCGTCCG
ACCATCGATATCTATTCATCTCCGTTTAATCTATATCCTACTGTTATTAAACACATTTCC
TATACTATCATGATGTGTCTTTGGGCTCTAGGGATCATATCTACCCACCTATCTAATCTG
ATTGGGTCATCACTTATTAATATACTACAGTGAATCAAGGCTCATCTAGCCTATCTGTCC
TCGGCTTACTATTCCGTCACCCAGAGTACCACCGAACGATGTCGGCCTATCCTCTAATCA
TCCTATCAATCTACTATCACAAGGTGCATCAATTCTACGTCGTTCTATCCAATCGAATCC
GGTCCATACCAATCTCAGTAGCTCCGACATTATTGACACTGTTAGGATCCCGTCGGTCAC
GTCCGTTCGGCTTCACCTTCCCAGCCTTAGTTGCCAGGCCTTAATCTAATCCTAGCTCCT
TATAATCTATATGGATTCTAGTCATATAACGCTAGGAAGATTAACGACTCCCGCTATTTA
CTACCCGATCGGTACGTCATCACACTACTGCCAGTGTATTTCTATTGGAAACCCTAACTC
CATTCTACTATGGTTAAATAAGAGTGGGTTCCTATGGATTAAAGCTCTAGTGTGCTCTTC
CTATGGTACTCATATCTCCTTCCTAAATTACTTACTCAAACACCTCCTTAAGCCAAATTC
TAGAGATATAATAAGTCAAATTCTATAGGGGTTTCTAACCAATTTAGTAGATCTATAACT
TACTTATCCCATAGGTTTCTAACTTACAACTTAGTCCTATAGGGCTTGATTTATTATATA
CAAGATAACTCACTCTATAAGCTTTGCTCACACATCATCTCACACCAATATATACCAAAA
TAGCTCTCAAAAGGATTTGACTCAACACCCCTATGGGATATCATCTAAGTCATCTAATTT
AACTAATATTTCTATTACATGGGCTAGAGTAGGTCTCTTTCAATCAATCATGCACCCATT
CCAAAAGTCTAG
>lcl|comp10877_c0_seq2 len=1160 path=[6037:0-34 11677:35-40 11683:41-46 1200:47-73 1227:74-108 1262:109-1159]
CTCATAGAGAGATTCGTCATCTAGGGAACAATGCAAATGCACACTAAATGAGTTAATTAA
ACATCCAATTATCACCATTAAGCAAGTCAAAATCAATCTAGAGCATTCCATGTGTATGCA
TAAGTTGGAAGTTAGAAAACCTTACCTGGAAGCCCTTCTGAGTACCTTAAAAAACTATAA
AAACTATCTAATCAAGGCAATTAATATAATCTCTAGAATTAATTGTAATTAGAAATCAAG
CTTAAGTCCTAAATATAAAACTAGGGCAAATATAATTATAAGTTAATCCAAGTCCTTATC
AAGTCCTAGTGAATCAAATTTTCAGTCAAGCTAAATCCTCAAAATTAAATATGGAATTAT
GTCAAGGTCAAGGCTTAGTCAGCTTATAATGGTCCTAGGTCTAGTCTAAGTCCTAGGGAA
AAAAAAGAAAGAAGAAAAAAACTAAAAAAACAAGTCAAAACTCATTATAGTGGAAAAATA
I have checked a few of my IDs manually and they are indeed in my database. Can anyone tell me what I am doing wrong? Or suggest another approach?
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Old 01-18-2012, 11:17 PM   #17
arvid
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Remove the leading ">" in your identifiers - it is not part of the ID, but a part of the FASTA format...
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Old 02-08-2012, 06:28 PM   #18
Volklor
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Question fastacmd gives errors

Your response to Anna is almost helpful to me...I have been using perl to extract seqs, but a one-liner, if it works, will be so much more efficient! However, when I tried to use fastacmd, and also blastdbcmd, I got an error for each entry in my query list, like this:

$ fastacmd -d contigs -i fastacmdtest.txt -o cp_contigs.fa
[fastacmd] ERROR: Entry "NODE_21_length_493_cov_13.705882" not found
[fastacmd] ERROR: Entry "NODE_75_length_1153_cov_20.143105" not found
[fastacmd] ERROR: Entry "NODE_2130_length_836_cov_4756.417480" not found
[fastacmd] ERROR: Entry "NODE_2409_length_1402_cov_21.002140" not found
[fastacmd] ERROR: Entry "NODE_2859_length_955_cov_1013.558105" not found

I know these entries are in my db because I copied them directly from the file from which I created the db in order to test the command. The test file looks like this:

NODE_21_length_493_cov_13.705882
NODE_75_length_1153_cov_20.143105
NODE_2130_length_836_cov_4756.417480
NODE_2409_length_1402_cov_21.002140
NODE_2859_length_955_cov_1013.558105

I just re-read the post above from kmcarr about indexing using makeblastdb. I used formatdb, so does the same issue apply there? Any idea what I'm doing wrong?

Last edited by Volklor; 02-08-2012 at 06:47 PM.
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Old 11-06-2014, 06:16 AM   #19
Hazel_Tan
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Default Extract contigs

Quote:
Originally Posted by kmcarr View Post
Anna,

You can do this yourself and it would be a good learning exercise, but since you have already made a BLAST database of the contigs, NCBI has kindly provided tools for doing exactly what you want.

Create a text file of the contig IDs you want to extract, one ID per line, no other information in the file. Be careful to use the same IDs as BLAST for your contigs. We'll call this file "myContigList.txt".

The command to use depends on whether you are using the old school (C-Toolkit) BLAST or the new BLAST+. These ancillary commands should have been installed when you installed BLAST

Old school use the command "fastacmd"

Code:
$ fastacmd -d myBlastDBName -p protein -i myContigList.txt -o myHitContigs.fasta
You can omit the '-p protein' and let the command guess the DB type.

For the new BLAST+ distribution use "blastdbcmd"

Code:
$ blastdbcmd -db myBlastDBName -dbtype prot -entry_batch myContigList.txt -outfmt %f -out myHitContigs.fasta
Again you could omit the '-dbtype prot' and let the program guess. The -outfmt %f tells the program to output sequences in FASTA format; you could also omit this since this is the default output format.


Hi kmcarr,

I would like to ask I used local blast+ to blast my own genome sequence with a query protein and also query nucleotide. My genome sequence i make it as subject instead of makeblastdb and the command is like this
Quote:
$tblastx -query /home/hazel/Dekstop/heterobasidion.fasta -subject /home/hazel/Dekstop/Gano.fasta -out tblastx_Result.txt -outfmt 1
My own genome sequence contain 4000 contigs after blast I have 500 contigs which hits the query. What should i do to extract those 500 contigs out of 4000 contigs?
Thank you so much. =)
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Old 02-16-2016, 11:55 AM   #20
mgallo2
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Default Looking for some help

Hello all-

Brand new to this site, but think this is the right form to seek help:
I'm trying to 'extract' nucleotide sequences from my results after running a local blastn against my local database. The output format right now is a standard "blast-looking" result page, but it's not easy to work with the results when I want to further compare sequences. (I have a gene- and will have genes- of interest. I want to see how they compare to the local database of my sequences, but then I want the results in FASTA format for further analyses and comparisons). The help manual for local blast doesn't seem to have the answers I am looking for

Will gladly provide additional information if more is needed. I really thank anyone who is able to help!
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