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I have been trying to learn some basic DNA sequencing techniques and have been using BLAST as a reference. I thought that I was starting to get it, but then I cam across this:

enter image description here

It looks like it's saying that the match starts at 10835 and ends at a smaller index (10714). I tried clicking the link to view the full sequence, and I couldn't find the highlighted portion anywhere (not at 10835 or at 10776).

My question is: Where (in NR_046235.1) can I find the highlighted sequence? And what does it mean to say that the Sbjct starts at 10835 and ends at a lower index?

I can post my query if necessary. Thanks.

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Just to help you with the answers below - there are lots of online tools which allow you to generate the reverse complement of a sequence if you have difficulty 'seeing' this, e.g. bioinformatics.org/sms/rev_comp.html –  Alan Boyd Apr 30 '13 at 17:24
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@Alan No need for that, a simple console command does the same for you: (tr ACGT TGCA | rev) <<< 'GATTACA' –  Konrad Rudolph Apr 30 '13 at 19:44
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2 Answers

up vote 4 down vote accepted

Just to flesh out Daniel Standage's answer a bit, remember that a genome is double stranded with one strand being complementary to the other. Genes can be found on either strand, the two are equivalent biologically. However, sequencing projects choose one of the two strands (randomly) and call it the plus (+) strand and then save all sequences with respect to that strand. This means that sometimes the genomic sequence that you download from a database might be the complement of the actual sequence you are looking for.

BLAST and similar algorithms take this into account and will compare your query sequence to both strands. If your query hits the target sequence on the - strand, BLAST will give a a result like what you have obtained, where the start of the HSP is lower than the end. This is because RNA translation (though not DNA transcription) goes in a 5' to 3' direction, on both strands. We therefore "read" genes in that direction. The coordinates are always with respect to the + strand since we always consider DNA in a 5' to 3' direction, this means that its start position will appear to be after its end position. This is better explained using an image:

enter image description here

In the image above, the blue line represents your BLAST hit. It stretches from position 10 to position 40 (with respect to the + strand). However, since it is on the minus strand, BLAST will show it starting at position 40 and ending at position 10.

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Thank you. Very helpful. –  Eric Apr 30 '13 at 19:30
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Note that the query sequence you have provided matches the minus strand of your target sequence. That means not only that the target sequence will be reversed (as you have noted), but it will also be complemented as well. So in the GenBank record, you should not look for the sequence CCGACCGA... starting at position 10835, you should look for the sequence ...TCGGTCGG ending at 10835.

ccgaccga...    minus strand
||||||||
bbɔʇbbɔʇ...    forward strand

The sequence in the GenBank record contains spaces, so that complicates finding your sequence using the browser's "Find" function, but if you use the position indices at the beginning and end of each line of sequence, you will be able to find the reverse complement of the matched sequence reported in the BLAST results.

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Thank you for this explanation! I accepted the answer by @terdon because it was slightly more beginner-friendly. –  Eric Apr 30 '13 at 19:29
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