Biol 312 L Molecular Biology Lab

DNA Sequencing Overview

DNA sequencing involves the determination of the sequence of nucleotides in a sample of DNA. It is presently conducted using a modified PCR reaction where both normal and labeled dideoxy-nucleotides are included in the reaction mix. The dideoxy-nucleotides lack the OH at their 3' carbon atom and are labeled with fluorescent dyes (each nucleotide has a different color). During PCR, mostly normal nucleotides are added to the DNA template. However, when a labeled nucleotide happens to add to the growing DNA strand, chain elongation stops because there is no 3' OH to which the next nucleotide can be attached. Hence, the dideoxy method is also called the chain termination method. At the end of the sequencing reaction, the fragments with fluorescent termini are separated by length from longest to shortest using a polyacrylamide gel (either a big thin slab gel or a narrow capillary tube filled with gel solution) that is scanned with a laser detection device. As each band moves past a viewer, the laser excites the dye, and the color of fluorescence is read by a photocell and recorded on a computer. A computer program evaluates the resulting histogram of fluorescence peaks and calls the nucleotides in the order they passed through the viewer. Interpreting Sequencing Results The computer output for a sequencing run consists of chromatogram (typically a sequencing facility will email you a file) that can be opened in one of many free DNA sequence viewers that are available on the web. We will be using a program called FinchTV. When viewing chromatograms, there is some ambiguity at various sites along the DNA sequence as the sequencing machine that is used to call the bases cant always precisely determine what nucleotide is represented by either a broad peak or a set of overlaying peaks. In such cases, a letter other than A, C, G, or T is recorded, most commonly N. When you obtain a sequence you should proofread it to ensure that all ambiguous sites are correctly called and determine the overall quality of your data. At the very minimum you should proofread and evaluate the quality of your sequence prior to uploading it to NCBI. If your sequence is suboptimal, you want to redo the sequencing or have both a forward and reverse sequencing reaction done and compare your two results to alleviate any possible miscalls.

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Biol 312 L Molecular Biology Lab Assignment: Viewing your Sequence and Analyses Open the Finch TV program. (Finch TV folder is on desktop, open it and hit the icon that resembles a TV). You will be directed to drag a sequencing file into the screen. Drag in your sequencing file. The first step is to examine your sequence from beginning to end by using the horizontal and vertical scroll bars. Good sequence generally begins roughly around base 20, and is represented by tall distinct peaks that have little overlap. Poor reactions will result in low or multiple peaks as illustrated below (very typical of beginning or end of a reaction).

Beginning of Sequence

End of sequence

At what base (indicated by the numbers at the top of the sequence) does your sequence become of high quality? (Note: some will not be of high quality anywhere, if this is the case get another sequence or compare a labmates)

How long is your sequence? Does the quality deteriorate towards the end? Do you find a stretch of AAAs at the end? (These AAAs are added by the Taq polymerase and are non-template derived.)

The second step is to check your sequence for ambiguity by searching for places where N is the base call instead of an A, C, G, or T. For example, in the case shown below, the correct nucleotide was likely a T. Do you find any instances where the N designation might not be appropriate? Also examine areas where two bases are superimposed on each other. Can you pick only one?

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Biol 312 L Molecular Biology Lab Look at the overall quality of the sequence, do you find that you have nice peaks that are easy to score? Does it appear that more than one product was sequenced?

Third step: determining homology. In other words, is your sequence similar to any other published sequences and if so, to what degree? This can be accomplished using BLAST, a program supported by the National Center for Biotechnology Information (NCBI). In Finch TV, select from the File dropdown menu the command export FASTA sequence and save the export onto the desktop. Open the exported file (you may need to open this up in the notepad program). You should see your sequence as a string of letters, copy the sequence and do a BLAST search at: http://www.ncbi.nlm.nih.gov/BLAST/cgi In the Basic Blast (left) section of this page, select nucleotide BLAST. Paste your sequence in the Enter Query Sequence box. In the Choose Search Set database, select other which includes all bacterial sequences. The drop-down menu should now say Nucleotide collection (nr/nt). You do not have to change any other parameters.

1) sequence in Fasta format

3) choose these 2) alternately you can upload your .seq file from the desktop

Click the Blast! button at the bottom to submit your sequence data.
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Biol 312 L Molecular Biology Lab

This screen will come up next. Finally (sometimes after a lengthy wait), a new window will appear showing any hits your sequence made. The results will be color coded and annotated so that you can either click on the horizontal homology bar(s) or you can simply scroll down the page.

many seq. with high homology

The bars show what places along your sequence are similar to other published sequences; the colors indicate how many bases were involved in homology determination. The scores indicate how much homology occurred for particular hits. Clicking on a score or bar in the graphic representation will take you to the section of the results page that describes a sequence that is similar to yours.

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Biol 312 L Molecular Biology Lab Clicking on a gi link at the beginning of any line will take you to the GenBank accession page for a sequence showing similarity to yours. There you can find a wealth of information about the published sequence to which yours showed some homology.

Fourth step: examine the proposed identities of your sequence. Ignore any uncultured bacterium because it does not designate an identity. Look for high query coverage, low E value (chance of random match) and high max identity scores. In this case, many instances of Enterococcus faecalis came up. Selecting the first Enterococcus sequence outlined in the box in the above figure (genbank# EU285587.1) will take you to the comparison between your query and the match. The majority of discrepancies are sequence gaps, with a few misalignments.

one misalignment several sequence gaps

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Biol 312 L Molecular Biology Lab Fifth step: now go back to YOUR sequence, check these areas, and decide if you would change anything. If you do change anything on your chromatogram, then go back and re-BLAST your sequence. Did any of the identity values change because of your corrections? What are the old and new values?

How does your sequence compare to its best match(es)? Fill out the BLAST results in the Table, then access the Ribosomal Database. BLAST RESULTS
NCBI Best match (Genus species) Your Sequence Alternate #1 Alternate #2 % Identity Organization where sequence was uploaded

RDB RESULTS
RDB Best match (Genus species) Your Sequence Alternate #1 Alternate #2 % Identity Organization where sequence was uploaded

To access the 16S rRNA database: http://rdp.cme.msu.edu/

Choose Sequence Match

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Biol 312 L Molecular Biology Lab Here is the start page:

you can upload your .seq file from the desktop here is your sequence in Fasta format

Paste in sequence. Click Submit

Next, the status page comes up:

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Biol 312 L Molecular Biology Lab

Finally, the results appear:

select this

This is the bottom half of the screen. You can try different parameters if you want.

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Biol 312 L Molecular Biology Lab

:: Result Format
Each match result line contains six elements, from left to right,

A short ID used to uniquely identify the RDP sequence. A click will return the simple entry, including the sequence. The orientation of the query sequence when the match is performed. "-" means the query sequence has been reverse-complemented. A top match hit with "-" orientation usually indicates the query sequence is a minus strand. A similarity score. SeqMatch reports the percent sequence identity over all pairwise comparable positions when run with aligned myRDP sequences. (Comparable positions are aligned positions containing a base in both sequences). Note that the rank order may differ between S_ab and pairwise identity scores, but the top 20 S_ab scores will contain the closest sequence by pairwise identity about 95% of the time (Cole et al). If two sequences do not overlap, the similarity between these two sequences will be displayed as "?". A seqmatch score (S_ab). These are the number of (unique) 7-base oligomers shared between your sequence and a given RDP sequence divided by the lowest number of unique oligos in either of the two sequences. The number of uniquely occurring oligomers within a given sequence (Olis). If the same oligomer occurs more than once then they are counted only once; thus this number only approximately reflects the sequence length. Counting only unique oligos compensates somewhat for composition bias (for example, inserts tend to be GC-rich and it becomes very likely that the same GC-rich oligos occur several times; by counting these only once, this artifact becomes less severe). Full name. The definition line from the RDP distribution, often the same as Genus/species/string name and accno.

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Biol 312 L Molecular Biology Lab Did results from NCBI and Ribosomal Database at the Genus species level come out the same? Fill out the Tables on page 6.

If there was a discrepancy between NCBI and the Ribosomal Database project which would you suspect is generally more accurate? Explain why.

NOTE: ANSWER THE QUESTIONS ON THE HANDOUT AND ON THE LAST PAGES (PAGES 11-12). KEEP THE HANDOUT AND TURN IN THE LAST PAGE.

REFERENCES FinchTV; http://www.geospiza.com/finchtv.html National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/ Ribosomal Database Project; http://rdp.cme.msu.edu/

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Your Name: _______________________

Biol 312 L Molecular Biology Lab Date: ___________________

Sequence: _______ Bacteria Identity: _____________________ % Identity: ________

Answer the questions in your handout an on this sheet. 1a. At what base (indicated by the numbers at the top of the sequence) does your sequence become of high quality?

1b. How long is your sequence? Does the quality deteriorate towards the end? Do you find a stretch of AAAs at the end?

2a. Do you find any instances where the N designation might not be appropriate? If so, give the nucleotide number of the N base and the base that you designated to replace N.

2b. Look at the overall quality of the sequence, do you find that you have nice peaks that are easy to score? Does it appear that more than one product was sequenced?

5a. check the areas that contain mismatches or deletions, and decide if you would change anything. If you do change anything on your chromatogram, then go back and re-BLAST your sequence. Did any of the identity values change because of your corrections? What are the old and new values?

5b. How does your sequence compare to its best match(es)? Fill out the BLAST results in the Table, then access the Ribosomal Database. BLAST RESULTS
NCBI Best match (Genus species) Your Sequence Alternate #1 Alternate #2 % Identity Organization where sequence was uploaded

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Biol 312 L Molecular Biology Lab RDB RESULTS

RDB Best match (Genus species) Your Sequence Alternate #1 Alternate #2 % Identity Organization where sequence was uploaded

5c. Did results from NCBI and Ribosomal Database at the Genus species level come out the same? Fill out the Tables on page 7.

5d. If there was a discrepancy between NCBI and the Ribosomal Database project which would you suspect is generally more accurate? Explain why.

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