phred: over 30 or over 25?

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cjones
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phred: over 30 or over 25?

Post by cjones » Wed Feb 14, 2007 8:48 pm

I just want to double-check on one of our targets. According to the finishing checklist, we want a Phred score over 25 for all bases in the consensus, and over 30 for bases covered by a single clone.

A. Has that changed? My TA said that we were now shooting for a score of 30 or more across the board, but I wanted to verify this.

B. Am I right to think that multiple clones, but with reads in only one direction, should also require a Phred > 30? And what of single clones but read in both directions or with multiple chemistries?
Chris Jones
Assoc. Prof. of Biology
Moravian College
Bethlehem PA

cshaffer
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finshing standard

Post by cshaffer » Thu Feb 15, 2007 5:30 am

Our goal is to meet the standard used to finish the mouse genome.

Basically this is:
consensus has a score over 25 if covered by reads from both strands or covered by more than one chemistry and phred 30 for single stranded/single chemistry [ That is any number of reads in one direction all with the same chemistry and no reads in the other direction or with an alternate chemistry.]

So...
A. Has that changed?
No it has not changed.
B. Am I right to think that multiple clones, but with reads in only one direction, should also require a Phred > 30?
Yes the goal for these is Phred 30. However the exact wording on the mouse standard for these regions is phred 30 or above *or* "reviewed by an experienced finisher". So the policy of the GEP is to go have students go ahead and order oligo's with the goal of getting all regions to either double stranded or to a Q score of 30. However, at the end if a region cannot be brought up to standard the region should be tagged and should be listed in the final status report. Then when the project comes back here we can have an experienced finisher give it a review.
And what of single clones but read in both directions or with multiple chemistries?
You would not know this looking at individual projects but there are no single clone regions in any of these project. Based on my analysis of the whole genome assembly, very region is covered by multiple fosmid clones.

If you are asking about regions in which you have a single read in each direction, or just two reads with different chemistries in the same direction then you would still need to have every base in the consensus have a score of 25 or above.

kstemler
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Finding the Phred score

Post by kstemler » Tue Jan 29, 2008 3:30 pm

How exactly do we find the phred score? I thought in training that there was a window that we could pull up which gave the phred score along with other information, however my professor and I do not remember how to access the specific window in question and therefore the phred scores. Please help.

cshaffer
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determine phred scores

Post by cshaffer » Thu Jan 31, 2008 7:12 pm

There are several ways to see the phred score. The first is to simply click on any base in the aligned reads window. Consed will print in the tiny window at the bottom of the aligned reads window some information about that base including the phred score.

Also in the default configuration I believe phred 30 is the cut off for full white background on any base.

Finally you can use your search for low quality navigator. This will give you a list of all bases in the consensus that are below a score threshold.
The default value of this is any base less than 25 but you can change this cut-off score in the "General preferences" menu item which is under the Options menu on the main window.

I usually have the students use the navigator to screen for anything with 25 or less and tag any bases that are less than 30 but double stranded and therefore satisfy mouse standard. The tags work well because the student will usually come back to a given problem area several times and the tag will let the student know they have already checked a given region and do not need to duplicate effort

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