SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > Illumina/Solexa



Similar Threads
Thread Thread Starter Forum Replies Last Post
How do you specify error rate in BBduk adapter trimming? antifolate Bioinformatics 11 07-07-2016 03:00 PM
Odd Ray Error "Assembler panic: no k-mers found in reads." jazz710 Bioinformatics 0 01-22-2016 06:58 AM
Odd results with Sickle FASTQ trimming id0 Bioinformatics 0 04-19-2013 09:07 AM
An odd error message from Tophat Mark.hz Bioinformatics 6 01-02-2011 09:34 PM

Reply
 
Thread Tools
Old 01-16-2017, 08:25 AM   #1
mike123
Junior Member
 
Location: California

Join Date: Jan 2013
Posts: 8
Unhappy Odd error while trimming Solexa data with AdapterRemoval???

Hello all,

I am attempting to process a published Solexa RNAseq dataset, but I am running into some issues due to the base quality encoding.

***This is the code I am using to attempt to trim adapter sequences:

$HOME/adapterremoval/bin/AdapterRemoval --qualitybase solexa --file1 $raw_files_path/$input_filename_1 \
--file2 raw_files_path/$input_filename_2 --basename $input_filename --trimns --trimqualities --gzip \
--adapter-list $HOME/RNAseq/adapters_set1.txt

***and this is the error I keep getting.


Read 2 adapters / adapter pairs from '/mnt/home/username/RNAseq/adapters-set1.txt'...
Trimming paired end reads ...
Error reading FASTQ record at line 1; aborting:
Phred+64 encoded quality score is less than 0 (ASCII < '@');
Are these FASTQ reads actually in Phred+33 format? If so,
use the command-line option "--qualitybase 33"

See README for more information.

I am not sure what to do, as the software is detecting quality scores that are less than zero (indicating Solexa encoding), but refusing to process the data even though I have specified "--qualitybase solexa" (as recommended in the user manual).

Normally I use Trimmomatic for adapter trimming, but I have successfully used AdapterRemoval (https://github.com/MikkelSchubert/ad...terRemoval.pod) in the past on Illumina Hiseq data.

Please help!!!

Thank You!!!
mike123 is offline   Reply With Quote
Old 01-16-2017, 10:30 AM   #2
kmcarr
Senior Member
 
Location: USA, Midwest

Join Date: May 2008
Posts: 1,133
Default

Quote:
Originally Posted by mike123 View Post
Hello all,

I am attempting to process a published Solexa RNAseq dataset, but I am running into some issues due to the base quality encoding.

***This is the code I am using to attempt to trim adapter sequences:

$HOME/adapterremoval/bin/AdapterRemoval --qualitybase solexa --file1 $raw_files_path/$input_filename_1 \
--file2 raw_files_path/$input_filename_2 --basename $input_filename --trimns --trimqualities --gzip \
--adapter-list $HOME/RNAseq/adapters_set1.txt

***and this is the error I keep getting.


Read 2 adapters / adapter pairs from '/mnt/home/username/RNAseq/adapters-set1.txt'...
Trimming paired end reads ...
Error reading FASTQ record at line 1; aborting:
Phred+64 encoded quality score is less than 0 (ASCII < '@');
Are these FASTQ reads actually in Phred+33 format? If so,
use the command-line option "--qualitybase 33"

See README for more information.

I am not sure what to do, as the software is detecting quality scores that are less than zero (indicating Solexa encoding), but refusing to process the data even though I have specified "--qualitybase solexa" (as recommended in the user manual).

Normally I use Trimmomatic for adapter trimming, but I have successfully used AdapterRemoval (https://github.com/MikkelSchubert/ad...terRemoval.pod) in the past on Illumina Hiseq data.

Please help!!!

Thank You!!!
Mike,

"Solexa" quality encoding of Q+64 has not been used in several years (eons in Next Generation Sequencing time). Hell, nobody even calls it "Solexa" anymore; it is Illumina. Do exactly what the error message suggests (highlighted above in red) and use "--qualitybase 33".
kmcarr is offline   Reply With Quote
Old 01-16-2017, 10:34 AM   #3
GenoMax
Senior Member
 
Location: East Coast USA

Join Date: Feb 2008
Posts: 6,484
Default

@mike123: If that is truly "solexa" format data of a ripe vintage then you may want to recode it to currently illumina before doing adapter removal.
GenoMax is online now   Reply With Quote
Old 01-17-2017, 08:10 AM   #4
mike123
Junior Member
 
Location: California

Join Date: Jan 2013
Posts: 8
Default issue resolved - thanks

Thank you both for your suggestions. After evaluating the raw data with FASTQC and actually looking at the *.fastq file entries (which I should have done in the first place...), it appears that the actual encoding is in fact Phred +33, and not Solexa (https://en.wikipedia.org/wiki/FASTQ_format#Encoding)

Lesson learned, yet again, never take summary info from public datasets at face value...
mike123 is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 01:56 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO