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Old 11-10-2010, 05:41 AM   #1
sbberes
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Default Software to filter errors in fastq files?

Hello community,
In the most recent data I have from an Illumina GAII the individual flowcell lane seq.txt files have a few random errors in the fastq format as determined using the FastX toolkit. Can someone recommend software or a script which will run thru a fastQ format file and discard or parse out the reads that have inappropriate formats or quality values.
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Old 11-10-2010, 06:00 AM   #2
drio
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What specific binary are you running within FastX and what's the exact command? What kind of "random errors" the tool is reporting?
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Old 11-10-2010, 06:18 AM   #3
sbberes
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Drio,
As background I am running multiplexed tagged SE 76nt sequencing runs. For this run there are about 50 million reads being generated per lane. As a first step before parsing the the tagged reads, I run the individual lane data thru the FastX tool kit to get an overview of the run. I am getting the following error messages when running fastx_quality_stats:

fastx_quality_stats: Error: invalid quality score data on line 112913096 (quality_tok = "\KJY\a\\Y_a\_aaa^cca_a_cRK_^^J\\\^\_cc^aa_aYccc084#0/1"
sbberes@computer1:~/Desktop/M59_GAS/ln2$

fastx_quality_stats: Error: invalid quality score data on line 74052832 (quality_tok = ""
sbberes@computer1:~/Desktop/M59_GAS/ln3$

fastx_quality_stats: Error: invalid quality score data on line 48710608 (quality_tok = "TTTCTNATTGTCGTGAAGGCATGACAACAGTATCAAGTCAAAAAGTCGGAGGCGGAGTTAATGTTTTTAGAGTTCC"
sbberes@computer1:~/Desktop/M59_GAS/ln4$

fastx_quality_stats: Error: invalid quality score data on line 69545528 (quality_tok = "__faad^dcXOcZfdaYa_adccSY`__YadaaYdd^aWSaaSWWM^ggdgB"
sbberes@computer1:~/Desktop/M59_GAS/ln5$

fastx_quality_stats: Error: invalid quality score data on line 99033924 (quality_tok = "gYgagf_cfffdffc`W]`BB"
sbberes@computer1:~/Desktop/M59_GAS/ln6$

fastx_quality_stats: Error: invalid quality score data on line 22453904 (quality_tok = "``````U[[`BBBBBBBBBBBBBBBBBBBBBBBBd^dcB"
sbberes@computer1:~/Desktop/M59_GAS/ln7$

I figure the easiest thing to do is just throw out the individual reads that have either inappropriate format or quality value designations.
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Old 11-10-2010, 06:42 AM   #4
maubp
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You left out the command line you used - in particular what FASTQ variant is the data, and what did you tell fastx to treat it as (with the -Q option).

There are two interesting errors there, an empty quality line, and what could be a sequence line being treated as a quality line. Have you looked at these regions of the file to see if there is anything obviously wrong?
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Old 11-10-2010, 08:53 AM   #5
sbberes
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All,
First, thanks for the feedback. The command line is just: fastx_quality_stats -i input-file -o output-file. The data is in standard fastq with illumina v 1.3 quality scores. I have the latest version of the standalone, not running under galaxy, version of the fastx tool kit. I cannot find in the documentation or using -h (help) any -Q parameter for this command. No, I have not gone to these regions of the file to look at the data. This is becoming an occurrence that comes up often enough in the data that I am getting that I was looking for an automated/scripted solution to run just as a matter of course. I am not a programmer by training, so before going to the trouble of writing a script myself I wanted to see if it had already been done for me by someone else.
SBB

Here are the first few line of the file:
@HWUSI-EAS528_16:1:1:1002:940#0/1
NNNGCTTGAAAAAATTGTTGGTTTAGCACTTGAAGATNNNNNTGNGNTANAGGGAGTACNNNGTNNNTNNNNNGNN
+HWUSI-EAS528_16:1:1:1002:940#0/1
BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB
@HWUSI-EAS528_16:1:1:1040:934#0/1
NGACTTCGAAAATCTCATCTGCATCTGATTATCAGGTTCTTTTTTTGAAAAAAACACCCAAAGTTATCTCTTATGT
+HWUSI-EAS528_16:1:1:1040:934#0/1
BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB
@HWUSI-EAS528_16:1:1:1094:930#0/1
NGTACTTCTATGGAATTTTTTTGTTTAATTATGGTCACAACGGCTCTATTTTAATGCCCAAGGATGTCGTTGAACA
+HWUSI-EAS528_16:1:1:1094:930#0/1
BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB
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Old 11-10-2010, 01:56 PM   #6
maubp
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Quote:
Originally Posted by sbberes View Post
All,
First, thanks for the feedback. The command line is just: fastx_quality_stats -i input-file -o output-file. The data is in standard fastq with illumina v 1.3 quality scores. I have the latest version of the standalone, not running under galaxy, version of the fastx tool kit. I cannot find in the documentation or using -h (help) any -Q parameter for this command.
It isn't documented, yet
See http://seqanswers.com/forums/showthread.php?t=7596
Quote:
Originally Posted by sbberes View Post
No, I have not gone to these regions of the file to look at the data.
I think you will have to in order to solve this - or switch to a different FASTQ tool: If you try using EMBOSS seqret, BioPerl, Biopython etc to parse the FASTQ do you get a more helpful error message?
Quote:
Originally Posted by sbberes View Post
This is becoming an occurrence that comes up often enough in the data that I am getting that I was looking for an automated/scripted solution to run just as a matter of course. I am not a programmer by training, so before going to the trouble of writing a script myself I wanted to see if it had already been done for me by someone else.
SBB

Here are the first few line of the file:
Code:
@HWUSI-EAS528_16:1:1:1002:940#0/1
NNNGCTTGAAAAAATTGTTGGTTTAGCACTTGAAGATNNNNNTGNGNTANAGGGAGTACNNNGTNNNTNNNNNGNN
+HWUSI-EAS528_16:1:1:1002:940#0/1
BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB
@HWUSI-EAS528_16:1:1:1040:934#0/1
NGACTTCGAAAATCTCATCTGCATCTGATTATCAGGTTCTTTTTTTGAAAAAAACACCCAAAGTTATCTCTTATGT
+HWUSI-EAS528_16:1:1:1040:934#0/1
BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB
@HWUSI-EAS528_16:1:1:1094:930#0/1
NGTACTTCTATGGAATTTTTTTGTTTAATTATGGTCACAACGGCTCTATTTTAATGCCCAAGGATGTCGTTGAACA
+HWUSI-EAS528_16:1:1:1094:930#0/1
BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB
I added [ code ] and [ /code ] tags round the example to stop the forum displaying it wrong. This can be done with the # icon on the advanced reply box.
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Old 11-11-2010, 04:02 AM   #7
sbberes
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Smile fastq_cleaner.pl

All,
A colleague at the Broad, Brian Haas (a good guy), provided me with a perl script which he generated for cleaning up fastq files. It steps thru the input file parsing the reads into those which conform to fastq format and those which do not, provides a count of each, and outputs the reads in a good and a bad file. it has solved my problem.
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Old 11-11-2010, 11:26 PM   #8
simonandrews
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If it's useful to anyone this is a small script I knocked up when we had to process some fastq files which were corrupted during an FTP transfer. You can pipe data through it and it does some basic sanity checks to ensure that the file looks like valid fastq data. It will remove any entries which look broken and leave you just the good stuff.

Code:
#!/usr/bin/perl
use warnings;
use strict;

while (<>) {

  unless (/^\@/) {
    warn "$_ should have had an \@ at the start and it didn't\n";
    next;
  }
  my $id1 = $_;
  my $seq = <>;
  my $id2 = <>;
  my $qual = <>;

  if ($seq =~/^[@+]/) {
    warn "Sequence '$seq' looked like an id";
    next;
  }
  if ($qual =~/^[@+]/) {
    warn "Quality '$qual' looked like an id";
    next;
  }
  if ($id2 !~ /^\+/) {
    warn "Midline '$id2' didn't start with a +";
    next;
  }

  if ($qual =~ /[GATCN]{20,}/) {
    warn "Quality '$qual' looked like sequence";
    next;
  }

  if (length($seq) != length($qual)) {
    warn "Seq $seq and Qual $qual weren't the same length";
    next;
  }

  print $id1,$seq,$id2,$qual;


}
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Old 11-12-2010, 03:18 AM   #9
sbberes
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Simon,
Thanks for the script, corruption of the files during transfer is exactly what I think probably occurred.
SBB
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Old 09-07-2012, 09:09 AM   #10
tarias
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Im trying to use this perl script but I don't know who to pipe my input file through this and get an output file with the good fastq file.
Thanks,
Tatiana


Quote:
Originally Posted by simonandrews View Post
If it's useful to anyone this is a small script I knocked up when we had to process some fastq files which were corrupted during an FTP transfer. You can pipe data through it and it does some basic sanity checks to ensure that the file looks like valid fastq data. It will remove any entries which look broken and leave you just the good stuff.

Code:
#!/usr/bin/perl
use warnings;
use strict;

while (<>) {

  unless (/^\@/) {
    warn "$_ should have had an \@ at the start and it didn't\n";
    next;
  }
  my $id1 = $_;
  my $seq = <>;
  my $id2 = <>;
  my $qual = <>;

  if ($seq =~/^[@+]/) {
    warn "Sequence '$seq' looked like an id";
    next;
  }
  if ($qual =~/^[@+]/) {
    warn "Quality '$qual' looked like an id";
    next;
  }
  if ($id2 !~ /^\+/) {
    warn "Midline '$id2' didn't start with a +";
    next;
  }

  if ($qual =~ /[GATCN]{20,}/) {
    warn "Quality '$qual' looked like sequence";
    next;
  }

  if (length($seq) != length($qual)) {
    warn "Seq $seq and Qual $qual weren't the same length";
    next;
  }

  print $id1,$seq,$id2,$qual;


}
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Old 09-07-2012, 09:40 AM   #11
simonandrews
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Quote:
Originally Posted by tarias View Post
Im trying to use this perl script but I don't know who to pipe my input file through this and get an output file with the good fastq file.
Thanks,
Tatiana
If you save that script to a file called filter.pl and give it execute permissions (chmod 755 filter.pl) then you can either pass your data in as a filename, or via a pipe (which you'll need to do if you have a compressed file for example).

To get the output simply redirect the output of the program to a file.

So to process a normal fastq file you'd do

./filter.pl starting_file.fq > filtered_file.fq

For a compressed file you could do:

zcat starting_file.fq.gz | ./filter.pl > filtered_file.fq

Hope this helps
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Old 09-07-2012, 10:09 AM   #12
tarias
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I did both and getting erros dump in the screen.

mu-166002esktop pireslab$ ./filter.pl TA16.txt > filtered.txt
Seq AAGGGGATACATTAGTTACATTTATATATGAAAAATCGAGATCCGGTGATATAACCCAAGGTCTTCCAAAAGTAGA
and Qual hhhhhghhhhechhhhhhgghhhghhhhhhhchgeehh_hfghhhhhhhhhedhfhachdcfKdacfccceheWhc_fcf
weren't the same length at ./filter.pl line 36, <> line 4.
Seq TTCTATCTCAAACAATAGCCTATCAACATCAAGTTCATCTCCTAAATCAGCTAGTTCAAGAAGAGGTCTAGAATTC
and Qual hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhghhhhhhhhhfhhhhhghachehfcdhaaf_fa_bdeb\Z\[^
weren't the same length at ./filter.pl line 36, <> line 8.
Seq GGATANAGACATGGCAAAAACTCCATCTGAGGAAGAACTGTATCACCATTTGTTGAATGGGGTAACTTGGACTCCG
and Qual hhhhhffffB`b``bhhhhhhhhhhhhhhhhhhghhfghhhhfhfgggfgfehhghghgadghhdfffhahdfdddcffb
weren't the same length at ./filter.pl line 36, <> line 12.
Seq ATTTANTCAACTTCCTGGTGATTCTCCACCACTTTATGTATTTAAATCAAGCTTCTTACAAAGTGATTCATCCTGG
and Qual hhhhhffffBdeeeehhhhhhhhhhhhhhhhhhhhghhhhhhgghhhhhhhhhhhhhhhhhgfgghcbhgfgghchhchh
weren't the same length at ./filter.pl line 36, <> line 16.
Seq GGATCNTCACAATCAAGTATAAGGCTGATGGTCAGGTTGAGAGAAAGAAGTCAAGACTGGTTGCAAGAGGTTTTAC
and Qual hhhhhffffBeeeeehhhhhhghhhhhhhghhcghhhchhhhhcfhhgghhhghgff_eefWfdagfhhghghfchffd_
weren't the same length at ./filter.pl line 36, <> line 20.
Seq GAATCATCATTGAGTTTTGGGTTACTTAATGTATTTTAAAGTTTGGACAGAATATGTGTGATCGAGTGTTGAAAAT
and Qual ggggggdgggggcggggggggggaggggggcffgggdeggggcgfgggggfadf_faccfegegfafdafcfcfffffff
weren't the same length at ./filter.pl line 36, <> line 24.
Seq AATACAGGATTTCCAATCCTAGCAGGAAAAGGAGGGAAACGGATACTCAATTTAAAAGTGAGTAAACAGAATTCCA
and Qual hhhhhhhhhhhhhhhhhhghhhhhhghhhhfhhhhhhhhhhhghhhfhghhhhfhhhegeghdhhhhfhhceb`da_cac
weren't the same length at ./filter.pl line 36, <> line 28.
Seq AGAGANACCAGCATCCTCCTCCCCATCGTCCTCCTCCCCATCGTCCTCCTCCCCATCATGATTTTCATCTTCAACC
and Qual hhhhgdeeeBbbba`hhhhhhhhhhhhhhhhhfhfghhghhhhefgdegegffbghbe`cc_caRaaaaaddccdfcddd
weren't the same length at ./filter.pl line 36, <> line 32.
Seq GTAAGNTCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAA
and Qual dc_f^]UZZBc^cacffffffffffffffffffffffffffffffffffffffffffcfffffaffffeccacBBBBBBB
weren't the same length at ./filter.pl line 36, <> line 36.
Seq TGAAACTGGATCGCTTGGCTCGGTGGAAGCGAGGCTCAGTTGTTGATCGGTGGAAGCGAGGCTCGGTTGGTGATCG
and Qual hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhghhhhhghhhhghhghhhhfhhfcggadhgdfbeeaeee`dcdbd
weren't the same length at ./filter.pl line 36, <> line 40.
Seq CTTCCTTGTTTGCCTTACAGAAGAACAAGCTATCATCTGCAAATAGAAAATGAGAGATTCCAGGGCAAGCTCTTGC
and Qual hhhhhhhhhhhhhhhhhhhhhhhhhhhhghhhhhhghhhhhhhdhdefcfbbb``fchahgghehfdaahggddefdccf






Quote:
Originally Posted by simonandrews View Post
If you save that script to a file called filter.pl and give it execute permissions (chmod 755 filter.pl) then you can either pass your data in as a filename, or via a pipe (which you'll need to do if you have a compressed file for example).

To get the output simply redirect the output of the program to a file.

So to process a normal fastq file you'd do

./filter.pl starting_file.fq > filtered_file.fq

For a compressed file you could do:

zcat starting_file.fq.gz | ./filter.pl > filtered_file.fq

Hope this helps
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Old 09-10-2012, 10:30 AM   #13
sklages
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You should post the first few lines of TA16.txt ... maybe we can see some problem ..
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Old 07-25-2013, 12:57 AM   #14
aforntacc
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Hi all i had this error message while using tophat. please how can i solve this error problem, because look very stock at the this point. and i am new to tophat, linux etc.
i am using ubuntu on a vitural machine ubuntu, the fastq file is of phred scale with Q30 as score.
kindly help
thanks
[2013-07-20 04:11:39] Checking for Bowtie
Bowtie version: 2.1.0.0
[2013-07-20 04:11:39] Checking for Samtools
Samtools version: 0.1.19.0
[2013-07-20 04:11:39] Checking for Bowtie index files (genome)..
[2013-07-20 04:11:39] Checking for reference FASTA file
Warning: Could not find FASTA file seq.fa
[2013-07-20 04:11:39] Reconstituting reference FASTA file from Bowtie index
Executing: /usr/bin/bowtie2-inspect seq > ./tophat_out/tmp/seq.fa
[2013-07-20 04:11:45] Generating SAM header for seq
format: fastq
quality scale: phred33 (default)
[2013-07-20 04:11:51] Preparing reads
left reads: min. length=100, max. length=100, 63588062 kept reads (424 discarded)
right reads: min. length=100, max. length=100, 63000645 kept reads (587841 discarded)
[2013-07-20 05:17:15] Mapping left_kept_reads to genome seq with Bowtie2
[2013-07-20 13:05:46] Mapping left_kept_reads_seg1 to genome seq with Bowtie2 (1/4)
[2013-07-20 13:59:24] Mapping left_kept_reads_seg2 to genome seq with Bowtie2 (2/4)
[2013-07-20 15:06:18] Mapping left_kept_reads_seg3 to genome seq with Bowtie2 (3/4)
[2013-07-20 15:56:59] Mapping left_kept_reads_seg4 to genome seq with Bowtie2 (4/4)
[2013-07-20 16:49:52] Mapping right_kept_reads to genome seq with Bowtie2
[2013-07-20 23:01:03] Mapping right_kept_reads_seg1 to genome seq with Bowtie2 (1/4)
[FAILED]
Error running bowtie:
Saw ASCII character -93 but expected 33-based Phred qual.
terminate called after throwing an instance of 'int'
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Old 07-25-2013, 01:12 AM   #15
mastal
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Default Software to filter errors in fastq files?

Q30 isn't a quality scale.

You need to know what version of the Illumina software/quality encoding was used. The current version is Illumina 1.9. Illumina has been using the Phred+33 scale since version 1.8, but earlier versions used different quality encoding scales.

See
http://en.wikipedia.org/wiki/FASTQ_format
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Old 07-25-2013, 02:16 AM   #16
aforntacc
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ok fine i get it, in the report it says hi-seq 2000, base calling pipleline hiSeq control soft. v 1.4.5
but by default bowtie identifies the quality scale. however now i am piping the fastq file through the perl script from the link you suggested will see when the run is finished.
i am new may be that's why it hard like this.
thanks a lot
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Old 07-25-2013, 02:24 AM   #17
mastal
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Default Software to filter errors in fastq files?

Bowtie doesn't identify the scale by default, phred33 is the default
scale bowtie will use unless you specify that your files use a different scale.

But still, I don't think there are any quality encodings that would give you a value of -93.

And yes, we've all been there, things always seem more complicated at the beginning.
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Old 07-30-2013, 08:36 AM   #18
aforntacc
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hello guys
Please i saw this summary file in the tophat out folder
please what does it mean and why is it 64% its very low. i googled a bit but became more confused
what can i do to to improve the mapping. i used default setting of tophat.

Left reads:
Input: 63588486
Mapped: 41120473 (64.7% of input)
of these: 5143253 (12.5%) have multiple alignments (2 have >20)
Right reads:
Input: 63588486
Mapped: 38423206 (60.4% of input)
of these: 4773086 (12.4%) have multiple alignments (0 have >20)
62.5% overall read alignment rate.

Aligned pairs: 31409898
of these: 3418180 (10.9%) have multiple alignments
and: 24649 ( 0.1%) are discordant alignments
49.4% concordant pair alignment rate.

thanks
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Old 07-31-2013, 01:05 AM   #19
dpryan
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There are a few questions you'll need to answer before anyone can help you:
1) How long are the reads?
2) Have you quality trimmed yet?
3) What organism is this?
4) What reference did you use?
5) What version of tophat/bowtie was this?
6) What was the exact command line argument used to start alignment?
7) What sort of experiment was this from?
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Old 07-31-2013, 07:01 AM   #20
aforntacc
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Quote:
Originally Posted by dpryan View Post
There are a few questions you'll need to answer before anyone can help you:
1) How long are the reads?
2) Have you quality trimmed yet?
3) What organism is this?
4) What reference did you use?
5) What version of tophat/bowtie was this?
6) What was the exact command line argument used to start alignment?
7) What sort of experiment was this from?

ok i see
1 reads are 100bp. i did clearing of fastq file
2 no i did not and i dont know how honestly
3 organism plant
4 ncbi mRNA
5 BOWTIE 2
6 TOPHAT2 path to ref.fa path to fastq file A1 and A2
7 rnaseq.
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