DATE: EXPERIMENT NO:

DNA SEQUENCING

INTRODUCTION

DNA sequencing is the process of determining the precise order of nucleotides

within a DNA molecule. It includes any method or technology that is used to determine
the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of
DNA.Allan Maxam and Walter Gilbert published a DNA sequencing method in 1977
based on chemical modification of DNA and subsequent cleavage at specific bases. Also
known as chemical sequencing, this method allowed purified samples of double-stranded
DNA to be used without further cloning. The chain-termination method developed by
Frederick Sanger and coworkers in 1977 (for which he won his second Nobel Prize) soon
became the method of choice, owing to its relative ease and reliability. When invented,
the chain-terminator method used fewer toxic chemicals and lower amounts of
radioactivity than the Maxam and Gilbert method. Because of its comparative ease, the
Sanger method was soon automated and was the method used in the first generation of
DNA sequencers.

PROCEDURE

1) Set up reactions in AbGene 96-well plate using the recipe below

Ingredients Volume (µl)

BigDye Terminator Mix v1.1 1.0
Sequencing dilution buffer (5x) 1.5
Autoclaved ddH20 1.5
Primer (1µM) 1.6
Plasmid DNA + autoclaved ddH20 4.4
Total volume 10.0µl

2) Cover plate with a rubber seal (avoid using oil). Run reactions on PCR tetrad
machines using the hot-lid setting.

Thermal cycling program

1. 96°C 1 min
2. 94°C 10 sec

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3. 50°C 5 sec
4. 60°C 4 min
5. Repeat cycle (step 2 to 4) 29 times
6. 10°C Hold

3. Precipitation of BigDye Terminator v1.1 cycle sequencing reactions

Ethanol/EDTA/sodium acetate precipitation

1) Label a clean sterile eppendorf for each reaction; don’t attempt more than 12
samples.

2) Add 2µl of 125mM EDTA, 2µl 3M sodium acetate (pH 5.2), 10µl autoclaved
ddH20 and 50µl 100% ethanol to each tube. (Ensure EDTA and sodium acetate is
at the bottom of the tube).

3) Transfer 10µl of sequencing reactions to the labelled 1.5ml eppendorf and briefly
vortex.

4) Incubate at room temperature for 15 mins in the dark* (put in a drawer).

5) Spin at 13,000rpm for 15 mins. Orientate eppendorfs with the hinges outermost so
the location of the DNA pellet is known.

6) Set a P200 pipette to 80µl and aspirate off the supernatant, taking care not to
dislodge the pellet. If residual ethanol/EDTA/NaOAc drops are left behind,
carefully remove with as lint-free kimwipe (i.e. roll a corner to make a tissue
wick). Ignore drops adjacent to the DNA pellet.

7) Add 195µl 70% ethanol (pre-cooled at -70C).

8) Spin at 13,000rpm for 5 mins

9) Repeat step 6 using a P200 pipette set to 200µl.

10) Place eppendorfs upright in a rack with lids open, cover with a lint-free tissue and
air-dry at room temperature in the dark for 15 to 60 minutes or until dry.

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 *Dark conditions are recommended by Sigma due to the photosensitive BigDye.
However it is known not to be essential.

4. DNA SEQUENCING ON THE ABI SEQUENCER 48-CAPILLARY INSTRUMENT

This method assumes that you are using a 36cm array and the standard protocols.
AT YOUR BENCH
Prepare your plate. Re-suspend your precipitated sequence reactions in 10uL of Hi-Di
formamide. Plate out 5uL of this re-suspension and 5uL of Hi-Di formamide (i.e. a 1:2
dilution) onto an ABI 96-well optical reaction plate. Use 10uL ddH 20 in empty lanes,
rather than formamide. Each plate contains two 48-lanes runs; odd columns = run1 and
even columns = run2. See attached guide to row/column loading order. Cover plate with
clean rubber septa and ensure there are no bubbles at the bottom of wells by spinning in a
post-PCR salad spinner. Denature at 95°C for 3 minutes (in a PCR machine or hot block)
and then place on ice until you load it into the machine.

AT THE SEQUENCER
Re-start the SEQUENCER computer (password is ‘grouse’).
Click on Unified Data Collection; wait for the icons to switch from red to yellow to
green. Minimize.
Create a new Plate Record → open Plate Manager.
If you have an existing Plate Record template then edit this (plate name and sample
names) in Microsoft XL, import directly into Plate Manager (click ‘Import’) and proceed
to Run Scheduler (below). Otherwise click on ‘New’.
New Plate Dialogue box will open.
ID (type the name of your plate. This must be unique)
Name (type the name of your plate)
Application (Sequencing Analysis)
Plate type (96-well)
Plate sealing (Septa)
Owner name/Operator name (your initials)
Click OK
The Sequencing Analysis Plate Editor will now open.
Enter your Sample Names.
Select a Results Group. You have to create your own Results Group initially.
Select an Instrument Protocol ( SEQUENCER_POP7_StdSeq_BDTv1).
Select an Analysis Protocol (StdSeq_KB_BDTv1.1).
Instructions for creating/editing Results Groups, Instrument Protocols and Analysis
Protocols appear over page.
Fill down (CtrlD) so that all samples contain all necessary information.

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Export the Plate Record as a template (E:\AppliedBiosystems\UDC\DataCollection
\Data\PlateRecordTemplates). In the future this plate record can be edited in XL (plate
name and sample names) and directly imported into the Plate Manager.
Click OK.
Open Run Scheduler.
Click Search in the Input Stack pane → the Add Plate to Input Stack window opens.
Type of search → Advanced
Enter your search criteria, then click Search. Select your plate and click Add → Done.
Your plate will appear in the Current Runs pane of the Run Scheduler window.
Place your plate into a black tray and cover with a white clip-on cover. Place into the
SEQUENCER.
Click the green ► to run your plate.
Monitor your run in Instrument Status, Capillary Viewer and Array Viewer.
When your run is finished it will be dumped into the E drive (Applied
Biosystems/UDC/data collection/data). You must then copy this file to one of the lab
PC’s.
Use SeqScape v2.0 or Sequence Analysis v5.0 to analyse your data.

CREATING A RESULTS GROUP

Each user has their own Results Group which defines (i) how files & folders are named,
(ii) where files are saved and (iii) the type of analysis (i.e. sequencing).
To create an Results Group, select New from the drop-down menu in the Sequencing
Analysis Plate Editor, or click on the Results Group icon in the explorer pane.
The Results Group Editor will open.
General – enter the Results Group name and owner (you).
Analysis – click ‘Do autoanalysis’.
Destination – use the default file destination
(E:\AppliedBiosystems\UDC\DataCollection \Data). This folder will be emptied monthly
so always copy your folders onto Alfred (G:\).
Naming – in the upper pane (Sample File Name Format), under ‘Format’, select ‘Sample
name’, then ‘Plate ID’ and then finally ‘Capillary #’. Use the same procedure to label the
‘Run Folder’ as ‘Plate ID_Date of run_Run #’.
Click OK to save.
Your Results Group will now appear in the Sequencing Analysis Plate Editor.

CREATING AN INSTRUMENT PROTOCOL

The instrument protocol contains everything necessary to run the instrument. It is

comprised of a Dye-Set, which specifies the sequencing chemistry (dye-set E for BigDye

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v1.1 and dye-set Z for BigDye v3.1), and a Run Module, which specifies the capillary
array length (36cm or 50cm), type of run (i.e. standard verses rapid sequencing) and the
polymer (POP7).
To create or edit an Instrument protocol select New from the drop-down menu in the
Sequencing Analysis Plate Editor, or from within the Sequence Analysis Protocol
Manager.
The Protocol Editor will open.
Select a Run Module (e.g. StdSeq36_POP7).
Select a Dye-set (e.g. E-BigDyeV1).
Set Type to ‘Regular’.
Click OK to save.

CREATING AN ANALYSIS PROTOCOL

An analysis protocol contains all the settings necessary for analysis and post-processing.
It is comprised of a basecaller algorithm (e.g. KB) and a DyeSet/Primer (or Mobility) file
(.mob) that is specific to each combination of polymer and sequencing chemistry. To
create or edit an Analysis Protocol click on New from the Plate Editor drop-down menu,
or do the same from the Protocol Manager. This will open the Sequence Analysis
Protocol Editor. Under the General tab give the protocol a name and indicate your
preference of sequence file formats. Under the Basecalling tab select the basecaller
algorithm and the DyeSet/Primer file. The KB.bcp algorithm is preferred because it
provides quality values (QV) for each base and also recognises mixed bases. Use
DyeSet/Primer file KB_ SEQUENCER_POP7_BDTv1.mob. In the ‘Ending Base’
pane, you can also specify when you want basecalling to end. This is particularly
important if your sequence products are short. Under the mixed bases tab you can choose
to identify mixed bases (i.e. heterozygotes) and specify the threshold detection level.
Finally under the clear range tab you can instruct the protocol to trim low quality data
from the ends of your sequence automatically. Click OK to save your analysis protocol.

5. ANALYSING DNA SEQUENCES IN SEQSCAPE V2.0

Two applications are available for analysis of DNA sequence data generated by the
SEQUENCER capillary instrument. Sequence Analysis v5.0 is software that will perform
base calling of DNA sequences and enables individual DNA sequence chromatograms to
be visualized and edited. It is useful if you have small numbers of sequences. SeqScape
v2.0 is software that combines the initial base calling of DNA sequence data performed
by Sequence Analysis v5.0, with advanced sequence editing and alignment tools (e.g.
such as in Sequence Editor and Sequencher). Sequence Analysis v5.0 is therefore
redundant if you use SeqScape. This protocol provides a brief introduction to the
analysis of DNA sequences in SeqScape v2.0.

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Re-start a lab computer using the Windows 2000 operating system.
Double click on the SeqScape v2.0 software and login (TABLAB, password = grouse). If
you get a warning message “no disk in drive” press continue.

Before you can import and analyse your DNA sequence data you need to create a
Project template. This is comprised of a Reference Data Group, Analysis Protocol,
Analysis defaults and Display Settings.
Select Tools and open the SeqScape Manager
First, create a Reference Data Group ( = a known reference sequence to which your
sequences will be aligned and compared. Each user needs their own RDG).
Select the Reference Data Group tab → Click New
RDG Properties will open. Complete the tabs:
General – specify a name that is unique and has meaning
ROI – add one or more Reference Segments (= known reference sequences):
Click on Add Ref. Segment –prompts you to import a GenBank nucleotide (.gb) file
(recommended). Alternatively, click on Paste Ref. Segment – this allows you to copy-
and-paste a nucleotide sequence straight into the RDG. You may also define ROIs
(Regions Of Interest such as exons, introns), arrange them into layers, create links to a
library of known alleles and import specific NT (nucleotide) and AA (amino acid)
variants.
Click OK to save your RDG.

For standard sequencing runs using BigDye Terminator Mix v1.1 the Analysis Protocol (
SEQUENCER_StdSeq_POP7_BDTv1.1) and Analysis defaults
( SEQUENCER_POP7_BDTv1.1) already exist. If you ran a standard sequencing module
on the SEQUENCER using BDTv1.1 then proceed to the ‘Create a Project Template’
step below. If you’re using a different version of BigDye Terminator mix (v2.0, v3.0 or
v3.1), or doing either Rapid or Long-Read sequencing, you will need to create a new
Analysis Protocol and new Analysis defaults. See over the page for instructions.

To create a Project Template, select the Project Template tab and click New.
Give the template a name (i.e. the same as your RDG name)
Select your Reference Data Group, Analysis Defaults (e.g.
SEQUENCER_POP7_BDTv1.1) and Display Settings (use
‘DefaultDisplaySettings_v2’) from the drop-down menus (Note that the default display
settings should be OK for all types of sequence runs and all chemistries, but they can be
edited by selecting Properties in the Display Settings tab.
Click OK to save
Close SeqScape Manager

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Now you can create your New Project and import your samples
File → New Project
Select your Project Template
Specify a Project Name
Click New and your project will open.
Click File → Import Samples to Project (CtrlM)
The Import Samples window will open.
Locate your sample files in the explorer pane. You should have transferred these files
from the SEQUENCERpc E: drive to your own folder on G: drive (Alfred).
At this point you can either:
(a) Add Samples (adds all samples you highlight or all samples in the selected folder),
OR (b) if you have multiple samples from each of a number of individuals (e.g. forward
& reverse sequences), then you can group these samples into Specimens (= individuals).
To do this (1) click New Specimen, (2) highlight this specimen in the Samples to Add
pane, (3) then select just the samples for that specimen in the explorer pane, and (4) click
‘Add Samples’. (5) Repeat for each specimen. You will need to manually edit specimen
names in the Project Navigator after the samples have been imported. Alternatively you
can use the Auto Add feature if, within your sample names, text delimiters flank a
common specimen name.
Click OK when you have finished adding samples.
Your specimens & samples will appear in the Project Navigator pane in the main
SeqScape window (notice the presence of a red line through each specimen icon– this
indicates that they are yet to be analysed).
Click the green ► to analyse your samples. This should be fast.
SeqScape will perform basecalling, assign quality values to each base, identify mixed
bases (if you requested it to) and trim low quality data from the ends of your sequences. It
will also automatically assemble and align samples within specimens and construct a
consensus sequence for each specimen. Sequences will be automatically reverse
complemented, where necessary.

CREATING AN ANALYSIS PROTOCOL

Select the Analysis Protocol tab → Click New
The Analysis Protocol Editor will open. Complete the tabs:
General – specify a name that captures the application (e.g. Long-Read), polymer
(POP7) and sequencing chemistry (e.g. BDTv3.1).
Basecalling – select a basecaller and the appropriate DyeSet/Primer (i.e. mobility) file
from the drop-down lists. The recommended basecaller is KB.bcp, because it provides
quality values (QV) for each base and can identify mixed bases (heterozygotes). The
DyeSet/Primer file is specific for each combination of basecaller and sequencing

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chemistry. You can also indicate how the software should recognise the end of your
sequence fragment.
Mixed Bases – select mixed base detection and specify threshold (if required).
Clear Range – provide instructions for trimming low quality data.
Filter – use the default values
Click OK. (Note: you could export your Analysis Protocol from Unified Data Collection
[on the SEQUENCER computer] and import it directly into SeqScape v2.0 by clicking
Import rather than New above).

CREATING NEW ANALYSIS DEFAULTS

Select the Analysis Defaults tab → Click New
New Analysis Settings will open. Complete the tabs:
General – specify a name that captures the application (e.g. Long-Read) and sequencing
chemistry (e.g. BDTv3.1).
Sample – Select the appropriate Analysis Protocol from the drop-down menu.
Specimen – Click ‘Base call samples’.
Otherwise use the default values
Click OK

Reviewing your analysis

Before editing any of your sequences check to see if your analysis was successful.
In the Project Navigator explorer panel click open each specimen to determine if
samples have been successfully assembled into segments (local alignments).
If any samples could not be assembled they will appear under ‘unassembled’. This could
indicate a failed or poor quality sequence, or that the analysis settings are inappropriate.
There are two ways to check:
First, click on the unassembled sample in the Project Navigator panel.
View the Annotation tab. Signal intensity should be 50 → 1000 for each base. Values
<50 probably indicate too little template. Values >1000 may be corrected by reducing
injection time (Edit the Instrument protocol on the SEQUENCER and re-run).
View the Sequence and Electrophenogram tabs. Click the ‘Show/Hide sample QVs’
icon to view quality values. How does the sequence look?
Second, click Analysis → Report Manager
The Report Manager provides links to 9 reports! (quickly explore these)
Select Analysis QC Report
The Specimen Analysis pane indicates if Basecalling, Filtering and Assembly of each
specimen has been successful (Green box), partially successful (Yellow triangle) or
unsuccessful (Red octagon). Reasons for Yellow or Red outcomes are provided in the
Sample Analysis pane. Sometimes poor results can be corrected or improved by editing
the analysis parameters and applying them to the affected samples/specimens.

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If necessary edit your analysis settings for failed samples and re-analyse. Instructions to
do this are provided over the page.

Viewing and editing your data

When you are satisfied with the analysis you can edit your data
First, turn on the Audit Trail (keeps track of all the edits you make and why you made
them):
Select Tools → Options from the main SeqScape window
Select Authentication & Audit and click the Audit Trail On check box
In the Audit Reason pane you can specify reasons for making changes
Click OK to exit the Options dialogue box.

Next, click the Project icon in the Project Navigator. Your specimen consensus
sequences will appear, aligned to the Reference sequence. This is called Project View.
Variable sites in the alignment are highlighted as coloured lines on the reference
sequence. Your current position in the alignment is indicated by the rectangle over the
reference sequence.
Click the triangle (►) adjacent to one (or more, or all) of your specimens so that you can
view the assembled & aligned samples.
Click on the consensus sequence to view the electrophenogram (EP) snippets in that
region of the alignment.
To centre the EP snippet use CtrlZ.
To edit the EP snippet range open Display Settings ( ds ), click View and edit ‘EP range’.
Click the ‘Show/Hide sample QVs’ and ‘Show/Hide consensus QVs’ icons to view
quality value bars for each base. Blue bars (QV 25-50) indicate that there is low
probability (≤0.31%) of an incorrect base call at that position. Yellow (QV 15-24) and
red (QV 0-14) bars indicate a progressively higher probability of error. Note that you
should be most concerned with the specimen consensus QVs.
YOU SHOULD MANUALLY EDIT: (1) all low (red) & medium (yellow) QV bases, (2) all
known and unknown variants (i.e. all polymorphic base positions), (3) all mixed base
calls and (4) all ambiguous base calls. But you should visually check the whole
sequence!
Click on the ‘View column selector’ icon (this highlights a single base for easy viewing)
Use the TAB key to jump through the sequence to the next base that requires editing.
To specify Tab settings, select ‘Multiple…’ from the Tab jump to next drop-down menu
(top RHS). The Multiple Tab Jump Settings box opens. Select options from the list and
click OK.
To actually insert, delete or change a base simply highlight the position on the sample or
consensus sequence and change the base. Changes made to the consensus will be
reflected in all samples that make up that specimen. Edited base changes appear in

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lowercase to distinguish them from unedited bases. If Audit Trail is activated you will be
prompted for a reason for making each change.

Exporting your data

When you have completed editing you can export Sample Sequence files, Consensus
sequences and also Project alignments in a number of formats, including FASTA, SEQ
and AB1.
To export a specimen consensus sequence:
Select File → Export → Consensus Sequence, and then export.

HOW TO EDIT YOUR ANALYSIS PROTOCOL FOR RE-ANALYSIS

From within the SeqScape main project window, click Analysis → Sample Manager
Click Edit Analysis Protocol.
Edit Basecaller, DyeSet/Primer file, Ending Base settings, Mixed Bases settings, the
Clear Range and Filter settings. Click OK.
Select the samples in the Sample Manager that you wish to apply the new protocol to.
Click Apply Analysis Protocol.
Select your new Analysis protocol from the drop-down list. Click OK and then Apply.
Click the green ► to re-analyse your samples.
Check the Project Navigator and the relevant reports in Report Manager.

RESULT:

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