rpsL DNA sequence analysis protocol
1. Download and install ApE: http://biologylabs.utah.edu/jorgensen/wayned/ape/ . If the "official" link does not work, try this one: http://www.navinpokala.org/protocols.html
2. Go to rpsL DNA sequence data and download the compressed file that matches your sequence tube code. Uncompress.
3. Open the .pdf file. See whether you have a "good" read. You should see that most of it has something like this:
1. Download and install ApE: http://biologylabs.utah.edu/jorgensen/wayned/ape/ . If the "official" link does not work, try this one: http://www.navinpokala.org/protocols.html
2. Go to rpsL DNA sequence data and download the compressed file that matches your sequence tube code. Uncompress.
3. Open the .pdf file. See whether you have a "good" read. You should see that most of it has something like this:
4. If you have a "bad" read, go to good reads and pick a new compressed file.
5. Open the .ab1 file with ApE. In ApE, use File -> "New DNA from basecalls". This opens a window with the chromatogram sequence ("Q5.ab1 basecalls" for example)
6. Download the rpsL_PCR.seq file using "save link as file" option. Open it with ApE. In this file, the rpsL START codon and STOP codon are in LOWERCASE.
7. Go to Tools -> "Align Two Sequences"
8. In the window that comes up, select the top “window” to your .abi trace file (not the basecalls), and the other to rpsL_PCR, and click OK. Leave the other settings at the default values.
9. The alignment window should show your sequence on top (your sequence) aligned to the wild-type on the bottom. Mis-matches, including gaps, will be highlighted. Ignore all except those between the start codon and stop codon; the sequence is of poor quality at the start and end of a read.
10. Double-click on a base on the top sequence in the alignment window. This will take you to that base in the chromatogram. Confirm that the chromatogram matches the called sequence by your eye; ie: make sure the computer didn’t make a mistake! Repeat for all the differences, and note changes (base position and what you think it should be).
11. Find the correct start and codon in your sequence. In the wild-type gene, the total coding sequence length, including start and stop codons is 375 bases. Make the start and stop codons lowercase by highlighting the codon and Edit -> "convert to lowercase". Edit your basecalls sequence to match your observations in step 10. Save your basecalls file.
12. Highlight the entire coding sequence, including the start and stop codons (375 bases in the wild-type with no insertions or deletions). Go to ORFs -> Translate. Select 1 letter code, DNA none. Click OK. This will create a new window containing the amino acid sequence and header information (* = stop). Highlight and copy just the amino acid sequence.
13. Go to Blast.
14. Paste the amino acid sequence into the top “Enter accession number(s), gi(s), or FASTA sequence(s)” form of the blastp webpage.
15. Repeat step 12-14 for rpsL_PCR, pasting the amino acid sequence into the bottom “Enter accession number(s), gi(s), or FASTA sequence(s)” form
16. Click the BLAST button. After the website is finished aligning, a new page will appear. Scroll down to "Alignments" to see the sequence alignment. The middle line is the consensus sequence, with mismatches, gaps, and insertions indicated.
17. What type of mutation(s) do you have?
5. Open the .ab1 file with ApE. In ApE, use File -> "New DNA from basecalls". This opens a window with the chromatogram sequence ("Q5.ab1 basecalls" for example)
6. Download the rpsL_PCR.seq file using "save link as file" option. Open it with ApE. In this file, the rpsL START codon and STOP codon are in LOWERCASE.
7. Go to Tools -> "Align Two Sequences"
8. In the window that comes up, select the top “window” to your .abi trace file (not the basecalls), and the other to rpsL_PCR, and click OK. Leave the other settings at the default values.
9. The alignment window should show your sequence on top (your sequence) aligned to the wild-type on the bottom. Mis-matches, including gaps, will be highlighted. Ignore all except those between the start codon and stop codon; the sequence is of poor quality at the start and end of a read.
10. Double-click on a base on the top sequence in the alignment window. This will take you to that base in the chromatogram. Confirm that the chromatogram matches the called sequence by your eye; ie: make sure the computer didn’t make a mistake! Repeat for all the differences, and note changes (base position and what you think it should be).
11. Find the correct start and codon in your sequence. In the wild-type gene, the total coding sequence length, including start and stop codons is 375 bases. Make the start and stop codons lowercase by highlighting the codon and Edit -> "convert to lowercase". Edit your basecalls sequence to match your observations in step 10. Save your basecalls file.
12. Highlight the entire coding sequence, including the start and stop codons (375 bases in the wild-type with no insertions or deletions). Go to ORFs -> Translate. Select 1 letter code, DNA none. Click OK. This will create a new window containing the amino acid sequence and header information (* = stop). Highlight and copy just the amino acid sequence.
13. Go to Blast.
14. Paste the amino acid sequence into the top “Enter accession number(s), gi(s), or FASTA sequence(s)” form of the blastp webpage.
15. Repeat step 12-14 for rpsL_PCR, pasting the amino acid sequence into the bottom “Enter accession number(s), gi(s), or FASTA sequence(s)” form
16. Click the BLAST button. After the website is finished aligning, a new page will appear. Scroll down to "Alignments" to see the sequence alignment. The middle line is the consensus sequence, with mismatches, gaps, and insertions indicated.
17. What type of mutation(s) do you have?
