SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics

Similar Threads
Thread Thread Starter Forum Replies Last Post
Unmatched read name error when converting csfasta to fastq with solid2fastq.pl yksikaksi Bioinformatics 2 11-06-2011 09:18 PM
Problem converting solid2fastq with Barcode m_elena_bioinfo Bioinformatics 7 09-15-2011 08:17 AM
Has somebody noticed that BWA' little scirpt solid2fastq.pl has some fatal defeat? ryuky Bioinformatics 3 04-23-2011 07:26 PM
solid2fastq paired-end m_elena_bioinfo Bioinformatics 2 07-08-2010 09:11 AM

Reply
 
Thread Tools
Old 05-25-2010, 05:20 AM   #1
Martin R
Junior Member
 
Location: Germany

Join Date: May 2010
Posts: 7
Default Solid2FASTQ

Hello

I just got a question. I wanna align SOLID data with BWA and therefore transformed it in FASTQ files with the solid2fastaq.pl script. But the script does not take the whole sequence of the csfasta but excludes the first two bp. In my opinion it would make sense to exclude the first base, as it is not in color space but not two.

Here an example:
an entry of my csfasta files has following sequence
>1_3_123
T000111222333

and the output in the FASTQ file is a sequence with:
AACCCGGGTTT

By comparing the sequences it can be seen that two bp are missing:
T000111222333

AACCCGGGTTT

this is because of line 100 in the script delivered by BWA 0.5.7 in the method read1:
$_ = substr(<$fhs>, 2);
This funktions takes the sequence beginning from the 3rd position.

Is there any reason for this or is this an error ?


Thanks for answering
martin
Martin R is offline   Reply With Quote
Old 05-25-2010, 09:04 AM   #2
nilshomer
Nils Homer
 
nilshomer's Avatar
 
Location: Boston, MA, USA

Join Date: Nov 2008
Posts: 1,283
Default

Quote:
Originally Posted by Martin R View Post
Hello

I just got a question. I wanna align SOLID data with BWA and therefore transformed it in FASTQ files with the solid2fastaq.pl script. But the script does not take the whole sequence of the csfasta but excludes the first two bp. In my opinion it would make sense to exclude the first base, as it is not in color space but not two.

Here an example:
an entry of my csfasta files has following sequence
>1_3_123
T000111222333

and the output in the FASTQ file is a sequence with:
AACCCGGGTTT

By comparing the sequences it can be seen that two bp are missing:
T000111222333

AACCCGGGTTT

this is because of line 100 in the script delivered by BWA 0.5.7 in the method read1:
$_ = substr(<$fhs>, 2);
This funktions takes the sequence beginning from the 3rd position.

Is there any reason for this or is this an error ?


Thanks for answering
martin
This is very subtle but important property of BWA: 2bp are lost from each read for SOLiD data.

The first color encodes the adapter and the first base, and therefore is partly dependent on DNA not present in the underlying genome (the adapter). BWA trims the first color and adapter so you lose the first two bases (a 50bp reads is now 48bp). This is performed so when the color read is compared to the encoded reference (in color space), the colors from the read will match the color reference exactly (when there is no error). Including the first color (or adapter) would violate those rules. Nonetheless, you lose two bases from each read, which BWA should be able to include/recover during local alignment (Smith-Waterman/Needleman-Wunsch), but does not. Other software, like BFAST (I am the author), can use all the bases in the final alignment, so it is possible. You should bug the BWA author (lh3) to remove the 2bp base trimming as it is not going to be fixed by changing the solid2fastq.pl, but only through changing much of the BWA color space code.

<rant>
It also seems like SOLiD support is an afterthought of many alignment software authors. Such a shame, since it is a much more fun and challenging problem to try and properly align in color space.
</rant>
nilshomer is offline   Reply With Quote
Old 05-26-2010, 12:58 AM   #3
KevinLam
Senior Member
 
Location: SEA

Join Date: Nov 2009
Posts: 192
Talking

Quote:
Originally Posted by nilshomer View Post

<rant>
It also seems like SOLiD support is an afterthought of many alignment software authors. Such a shame, since it is a much more fun and challenging problem to try and properly align in color space.
</rant>
hahah agreed.. is <rant> a new BB code? ROFL
KevinLam is offline   Reply With Quote
Old 05-27-2010, 12:08 AM   #4
Martin R
Junior Member
 
Location: Germany

Join Date: May 2010
Posts: 7
Default

Thank you very much for that fast answer.
Now I understand the handling of neglecting the first 2bp.
Martin R 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 03:03 PM.


Powered by vBulletin® Version 3.8.6
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.