Question: +/- strand conventions for probe sequences
1
gravatar for eric.kern13
17 months ago by
eric.kern1370
United States
eric.kern1370 wrote:

Hi Biostars,
I recently followed [this question](Human Exome Capture Library Coordinates Download) in order to obtain sequences for exon-capture probes sold in Agilent kits. Column headers plus one line of the file looks like this:

    TargetID        ProbeID     Sequence        Replication     Strand  Coordinates

    mRNA|AL390972   A_36_B233385 <a string of 120 ACGTs>    NA      +     chr1:100111836-100111955

I want to know which way the probes are aimed: will a given probe amplify regions towards lower genomic coordinates or higher? Since I couldn't find any sort of readme file, I need help figuring out where the 3' and 5' ends are and how that relates to the coordinates. For a simple example, suppose I were to convert this 'drawing' into a file of the sort that Agilent publishes.

    coords:     1  4  7  10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70
    sequence:   AAAAGGGGAAAAGGGGAAAAGGGGAAAAGGGGAAAAGGGGAAAAGGGGAAAAGGGGAAAAGGGGAAAAGG
    probe:               5-AGGGGAAAA-3 ==>                                            
    probe on reverse strand:               <== 3-TTTCCCCTT-5                           
    probe on forward strand:                                  5-AAAAGGGGA-3 ==>       

    direction of replication: ==> or <==


​      
Should (a few columns of) the file look like this? This way, everything goes 5' to 3', and I've reverse-complemented the probe on the minus strand.

            sequence   coords    strand
            AGGGGAAAA  chr22:12-20  +
            AAAGGGGAA  chr22:33-41  -
            AAAAGGGGA  chr22:49-57  +

Thank you for your help.

pcr sequence • 621 views
ADD COMMENTlink modified 17 months ago • written 17 months ago by eric.kern1370

Generally the sequence itself is that of the probe, which will always be 5'->3' (as is the case for all nucleotide sequences). So just check if the strand matches that of the probe itself or what if detects accordingly. If you want to be absolutely sure then just ask Agilent.

ADD REPLYlink written 17 months ago by Devon Ryan60k
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