SEQanswers

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



Similar Threads
Thread Thread Starter Forum Replies Last Post
unmapped reads with ssaha2 sammy07 Bioinformatics 2 10-22-2012 02:23 AM
BWA - unmapped reads Adamo Bioinformatics 23 02-22-2012 09:12 PM
Velvet for unmapped reads leeht Bioinformatics 1 11-20-2010 04:39 PM
bfast for unmapped reads Protaeus Bioinformatics 2 11-17-2010 02:35 PM
What are the unmapped reads beelu Illumina/Solexa 1 09-09-2010 05:18 AM

Reply
 
Thread Tools
Old 11-23-2009, 09:34 AM   #1
bioinfosm
Senior Member
 
Location: USA

Join Date: Jan 2008
Posts: 482
Default % unmapped reads

Hi all,

For any solexa run, we see 60-80% passing filter reads, of which 70-90% map to the reference sequence. There are quite a few reads that map to adaptors and other random sequences.

Does anyone know of a resource to get an average for those passing-filter reads? that map to adapter, etc and are essentially noise and not useful. I remember it being mentioned in some review as well, but cannot recollect

thanks..
__________________
--
bioinfosm
bioinfosm is offline   Reply With Quote
Old 12-11-2009, 03:05 PM   #2
der_eiskern
Member
 
Location: California

Join Date: Jul 2009
Posts: 46
Default

i'm not sure what exactly you want ("an average for those passing-filter reads")

but i've also noticed often two-thirds of the 30-10% of unmappable reads are alignable if you can tolerate a higher error rate.

from cloning random fragments i've noticed that 1/10-1/100 of cloned fragments from a sequencing library will have a truncated adaptor or multiple adaptors. since i didn't get my adaptors from illumina directly this could reflect contamination of my modified IDT oligos. it varied between library preps using the same batch of adaptors.

can you not just make a fasta file of the adaptor and iteratively truncate your read during mapping to such a fast a file to estimate it? i'd be more interested in seeing what what doesn't pass filter looks like, any idea how to get that data from the pipeline?
der_eiskern is offline   Reply With Quote
Old 12-11-2009, 09:04 PM   #3
Xi Wang
Senior Member
 
Location: MDC, Berlin, Germany

Join Date: Oct 2009
Posts: 317
Default

Quote:
Originally Posted by der_eiskern View Post
from cloning random fragments i've noticed that 1/10-1/100 of cloned fragments from a sequencing library will have a truncated adaptor or multiple adaptors. since i didn't get my adaptors from illumina directly this could reflect contamination of my modified IDT oligos. it varied between library preps using the same batch of adaptors.
I am wondering what makes the sequencer not report the adaptor sequences and exactly the very beginning of what we want to sequence. Or it will be quite normal to sequence the tails of adaptors.

Quote:
Originally Posted by der_eiskern View Post
can you not just make a fasta file of the adaptor and iteratively truncate your read during mapping to such a fast a file to estimate it? i'd be more interested in seeing what what doesn't pass filter looks like, any idea how to get that data from the pipeline?
Those not passing the filtering usually are with low quality scores, which indicates that the base calling may be incorrect or it is hard to call bases.
__________________
Xi Wang
Xi Wang is offline   Reply With Quote
Old 07-04-2010, 09:15 AM   #4
aleferna
Senior Member
 
Location: sweden

Join Date: Sep 2009
Posts: 121
Default Solexa Sequencing of Paired Ends

Does anybody know what is the expected percent of adapter sequence that one would expect in a Solexa sequencing run?

I'm mapping this solexa paired end run and I had to mask away about 50% of the sample because it maps to the adapters. Is this normal? If so and we do this again, how would you minimize the amount of adapter sequence that you get in the library????

If it is not normal, is there any doc/spec that we can use to complain to the sequencing service and get it done properly?
aleferna is offline   Reply With Quote
Old 07-04-2010, 08:46 PM   #5
Xi Wang
Senior Member
 
Location: MDC, Berlin, Germany

Join Date: Oct 2009
Posts: 317
Default

Quote:
Originally Posted by aleferna View Post
Does anybody know what is the expected percent of adapter sequence that one would expect in a Solexa sequencing run?

I'm mapping this solexa paired end run and I had to mask away about 50% of the sample because it maps to the adapters. Is this normal? If so and we do this again, how would you minimize the amount of adapter sequence that you get in the library????

If it is not normal, is there any doc/spec that we can use to complain to the sequencing service and get it done properly?
It may depend on what you sequenced. RNA-seq? miRNA-seq?
__________________
Xi Wang
Xi Wang is offline   Reply With Quote
Old 07-05-2010, 12:10 AM   #6
aleferna
Senior Member
 
Location: sweden

Join Date: Sep 2009
Posts: 121
Default

just plain human DNA
aleferna is offline   Reply With Quote
Old 07-05-2010, 12:17 AM   #7
Xi Wang
Senior Member
 
Location: MDC, Berlin, Germany

Join Date: Oct 2009
Posts: 317
Default

I have no experience on plain DNA sequencing. But I think your problem may largely due to sample preparation.
__________________
Xi Wang
Xi Wang is offline   Reply With Quote
Old 07-05-2010, 12:35 AM   #8
aleferna
Senior Member
 
Location: sweden

Join Date: Sep 2009
Posts: 121
Default

But in what part, is that the main issue is that we provide the samples, but another lab did the Paired End preparation. If there was a problem in the paired end preparation we need to go back to this people and tell them that they made a mistake. Because its such an expensive test this can get really ugly and I don't have any reference to make the point...
aleferna is offline   Reply With Quote
Old 07-05-2010, 12:36 AM   #9
aleferna
Senior Member
 
Location: sweden

Join Date: Sep 2009
Posts: 121
Default

How much do you expect in RNA?
aleferna is offline   Reply With Quote
Reply

Tags
adaptor, noise, passing-filter, solexa, unmapped

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 06:40 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO