Hi David,

Your files appear to be of the Sanger FASTQ variant. As you have noticed, the 
info blurb provided by the Grooming tool provides information that should be 
utilized to confirm input types. While the 'Illumina 1.3+' FASTQ  format does 
encode scores using a different ASCII range, it is my understanding that the 
scripts provided by the manufacturer to create FASTQ formatted files were 
enhanced to write out Sanger encoded quality scores. 

The correct Grooming path for your data is Sanger --> Sanger.  Please let us 
know if we can provide further assistance.

Thanks for using Galaxy,

Dan


On Mar 21, 2011, at 9:42 AM, David K Crossman wrote:

> Hello!
>  
>                 I am fairly new to using Galaxy and have a question about the 
> FASTQ Groomer feature.  I have 4 RNA-Seq raw data files that were just 
> recently generated from Illumina’s NGS instruments.  I am aware that the 
> first step to perform in Galaxy is FASTQ Groomer to convert the format to 
> FASTQ Sanger.  I presume that I would choose Illumina 1.3+ in the “Input 
> FASTQ quality scores type” box.  However, if I look at the raw data reads, I 
> notice that Line 4 (which encodes the quality values for sequence in Line 2) 
> has values outside of the Illumina 1.3+ range (some of them fall into the 
> Sanger format.  I am enclosing the Quality Score Comparison figure along with 
> some of the raw RNA-Seq data): 
> Quality Score Comparison
> SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS
> ...............................IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
> ..........................XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX
> !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~
> |                         |    |        |                              |      
>                |
> 33                        59   64       73                            104     
>               126
>  
> S - Sanger       Phred+33,  93 values  (0, 93) (0 to 60 expected in raw reads)
> I - Illumina 1.3 Phred+64,  62 values  (0, 62) (0 to 40 expected in raw reads)
> X - Solexa       Solexa+64, 67 values (-5, 62) (-5 to 40 expected in raw 
> reads)
> Diagram adapted from http://en.wikipedia.org/wiki/FASTQ_format
> RNA-Seq raw data
> @HWI-ST156_294:7:1:1058:2165:0/1
> CACCAACTCACAGCCACTCCGTGAGGCCAGCAAGGCAAGAACATTCATCTC
> +
> HHHHFGGHHHGFHHFHHEGHC<GGGEB.EE9D?DDEEEE4FFFCBB/.C=D
>  
> @HWI-ST156_294:7:1:1184:2191:0/1
> CGTAAATCCATGTCTGACTTCTGGATAGCAAACACCAGCACCGCGTGGATG
> +
> EE;E=ECEEBE@EEEE=GBFGF/GFFC<FA;:@<8AEABB>A#########
>  
> @HWI-ST156_294:7:1:1018:2200:0/1
> NCTGATTAAGGATAATGAGTTTTTAGTAGAACTAATGATGTTATTCCTTGG
> +
> ###################################################
>  
> @HWI-ST156_294:7:1:1225:2217:0/1
> GTTTTTGACTACACAAAGCACCCTTCTAAACCAGACCATTCTGGAGAATGA
> +
> FFCEFFFE?FEBDC?987::,3:<-9145,DA<:C9;+?############
>  
>  
>                 As a test in FASTQ Groomer, I chose either the Sanger or 
> Illumina 1.3+ as the input quality scores type and these are the results I 
> got:
>  
> FASTQ Groomer on tn-read1 (using Sanger as input)
> 6.1 Gb
> format: fastqsanger, database:mm9
> Info: Groomed 45868679 sanger reads into sanger reads. Based upon quality and 
> sequence, the input data is valid for: sanger Input ASCII range: '#'(35) - 
> 'I'(73) Input decimal range: 2 - 40
>  
> FASTQ Groomer on tn-read1 (using Illumina1.3+ as input)
> 6.1 Gb
> format: fastqsanger, database:mm9
> Info: Groomed 45868679 illumina reads into sanger reads. Based upon quality 
> and sequence, the input data is valid for: sanger Input ASCII range: '#'(35) 
> - 'I'(73) Input decimal range: -29 - 9
>  
> Which one is right (I presume the Illumina 1.3+ one, but I can’t find any 
> sort of explanation)?  I noticed that the “input decimal range” had different 
> values (although they spanned the same length) in relation to which input was 
> chosen.  What would happen downstream in TopHat if Sanger was used instead of 
> Illumina 1.3+ for these files?  Is there any other reading 
> material/websites/etc… out there that might help me better understand the 
> quality score and which to use?  Any info/help would be greatly appreciated.
>  
> Thanks,
> David
>  
>  
> David K. Crossman, Ph.D.
> Systems Biologist/Analyst/Statistician
> Heflin Center for Genomic Science
> University of Alabama at Birmingham
> 720 20th Street South
> Kaul Room 420
> Birmingham, AL 35294-0024
> (205) 996-4045
> (205) 996-4056 (fax)
> David K. Crossman, Ph.D.
> Heflin Center for Genomic Science
>  
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