Thermo Fisher Scientific Using the ABI 373 BigDye Filter Wheel: ABI PRISM® 373 DNA Sequencer Owner's manual

Brand: Thermo Fisher Scientific

Model: Using the ABI 373 BigDye Filter Wheel: ABI PRISM® 373 DNA Sequencer

Type: Owner's manual

User Bulletin

ABI PRISM® 373 DNA Sequencer

February 16, 1998 (updated 06/2001)

SUBJECT: Using the ABI PRISM 373 BigDye Filter Wheel

Contents The following topics are discussed in this bulletin:

Introduction This service part will allow users of the ABI PRISM 373 DNA

Sequencer to take advantage of Applied Biosystems new

dichlororhodamine (dRhodamine)-based chemistries:

♦BigDye™ terminators

♦dRhodamine terminators

♦BigDye primers

Topic See page

Preparing to Use the ABI PRISM 373 BigDye Filter Wheel 3

Choosing a Sequencing Chemistry 3

Software Information 6

Creating a dRhodamine Instrument (Matrix) File 8

Making a Matrix from Matrix Standards 8

Making a Matrix from a Sample File 20

Sequencing Reaction Setup 21

Sample Loading Recommendations 22

Quick Start Guide for the ABI PRISM 373 BigDye Filter Wheel 23

Appendix A: Part Numbers 25

Appendix B: Dye/Base Relationships 26

Appendix C: Load Chart 27

Page 2 of 28 User Bulletin: ABI PRISM® 373 DNA Sequencer

To allow 373 Sequencer users to use the dRhodamine-based

chemistries, a new filter wheel is now available. The band pass

wavelengths of the old and new filters are listed in Table 1 below. The

dye/base relationships of the rhodamine-based and

dRhodamine-based chemistries can be found in “Appendix B:

Dye/Base Relationships” on page 26.

Emissions from the dRhodamine dyes (dR110, dR6G, dTAMRA, and

dROX) are red shifted in the spectrum, which makes them

incompatible with the originally installed 373 Sequencer filter set A.

This filter set was designed to work with the standard dye primers or

rhodamine terminators. Emission spectra from the rhodamine and

dRhodamine dyes are shown in Figure 1 below.

Figure 1. Emission spectra from the rhodamine and dRhodamine dyes.

Note that the gray bars represent the emission maxima of the four dyes in

each dye set.

Note The unused filter in the BigDye filter wheel is 555 nm, replacing the

545-nm filter. This filter is not used for dRhodamine sequencing applications.

The advantage of using the dRhodamine-based chemistries with the

BigDye filter wheel is that the four dyes have narrower emission

spectra leading to less spectral overlap (see Figure 1). This translates

into reduced background noise and cleaner signals. In addition, the

BigDye chemistries, with their single-molecule energy transfer

Table 1. Wavelengths of Old and New Filters

Filter Set A Color BigDye Filter Set A

531nm Blue 540 nm

560 nm Green 570 nm

580 nm Yellow 595 nm

610 nm Red 625 nm

User Bulletin: ABI PRISM® 373 DNA Sequencer Page 3 of 28

system, produce 4–5 times more signal to noise than the existing

chemistries. The BigDye terminator and dRhodamine terminator

chemistries produce data with significantly more uniform peaks. This

improves basecalling accuracy and leads to longer read lengths.

IMPORTANT When the BigDye filter wheel is installed, the instrument

will still indicate it is running filter set A with the old wavelengths. The kit

contains a label that lists the new wavelengths and indicates the instrument

has been upgraded to the BigDye filter wheel. At install, the service engineer

will place the label on the instrument.

IMPORTANT When the BigDye filter wheel is installed, the FS rhodamine

terminators and FS dye primers will no longer be compatible. In addition, you

cannot perform GeneScan® software analysis with the BigDye filter wheel

because the emissions from the GeneScan software dyes are not collected

efficiently.

Preparing to Use

the ABI PRISM 373

BigDye Filter

Wheel

To successfully use the new filter wheel and new sequencing

chemistries, read “Software Information” on page 6 and “Creating a

dRhodamine Instrument (Matrix) File” on page 8. Before results can

be analyzed, these important setup steps must be performed.

Choosing a

Sequencing

Chemistry

The generally recommended chemistries are BigDye terminator and

BigDye primer because of their optimal signal-to-noise

characteristics. The choice between terminator and primer chemistry

depends on the application. See Table 2 on page 4 for chemistry

recommendations for different applications.

The dRhodamine terminators are useful for templates with long

homopolymer (> 25 bases) stretches or templates with GT rich motifs.

However, the dRhodamine terminators produce weaker signals than

the BigDye chemistries; more of the sample must be loaded to ensure

adequate signal is available. This is especially important for the

ABI PRISM 373 DNA Sequencer with the XL Upgrade (373XL)

because the 373XL has higher lane density, which requires stronger

signal strength for optimal performance.

Page 4 of 28 User Bulletin: ABI PRISM® 373 DNA Sequencer

R Recommended

S Satisfactory

N Not Recommended

Table 2. ABI PRISM 373 with BigDye Filter Wheel/310/377

Chemistry Recommendations

BigDye

Terminators

dRhodamine

Terminators

BigDye

Primers

DNA Sequencing Application

de novo

Sequencing High Throughput R S R

de novo

Sequencing Mid to Low

Throughput

RSS

Comparative Sequencing

(germline mutations 50:50

heterozygotes)

RSR

Comparative Sequencing

(somatic mutations 30:70 heterozygotes)

SNR

Comparative Sequencing

(somatic mutations 10:90 heterozygotes)

NNR

DNA Sequence Context

G-C rich > 65% R S S

A-T rich > 65% S R R

G-T rich (repeats) N S R

Homopolymer A or T > 25 bp N R R

Te m p l a t e

Plasmid (< 15 kb) R R R

M13 R R R

BAC, Cosmid, Lambda, XL PCR R S S

Bacterial genomic DNA R N N

PCR Amplicon R R R

PCR Amplicon (heterozygous 50:50) R S R

PCR Amplicon (heterozygous 30:70) S N R

PCR Amplicon (heterozygous 10:90) N N R

User Bulletin: ABI PRISM® 373 DNA Sequencer Page 5 of 28

Suggested Gel

Types

The BigDye filter wheel has been tested with the following gel types:

♦5.75% Long Ranger, 34-cm WTR

♦4.75% 19:1 acrylamide:bis acrylamide, 34-cm WTR

♦5% Long Ranger, 48-cm WTR

♦4% 19:1 acrylamide:bis acrylamide, 48-cm WTR

♦5% 29:1 acrylamide:bis acrylamide, 34-cm WTR

Mobility files were developed using these gel types. However, mobility

files may work for other gel types:

♦5.75% PagePlus, 34-cm WTR

♦5.25% PagePlus, 48-cm WTR

Page 6 of 28 User Bulletin: ABI PRISM® 373 DNA Sequencer

Software Information

Required Files Before samples can be analyzed, two files that interface with the

software must be in place: a dRhodamine instrument (matrix) file and

dye set/primer (mobility) files for the BigDye filter wheel. Instructions

for creating the dRhodamine instrument (matrix) file begin on page 8.

Instructions for installing the mobility files are given in “Installation of

Mobility Files” on page 7.

The mobility files are:

373BDP Rev Use with ABI PRISM® BigDye™ Primer Cycle

Sequencing Ready Reaction Kit with AmpliTaq®

DNA Polymerase, FS, M13Rev.

373BDP -21M13 Use with ABI PRISM® BigDye™ Primer Cycle

Sequencing Ready Reaction Kit with AmpliTaq®

DNA Polymerase, FS, -21M13.

373dRhod Use with ABI PRISM® dRhodamine Terminator

Cycle Sequencing Ready Reaction Kit with

AmpliTaq® DNA Polymerase, FS.

373BDT Use with ABI PRISM® BigDye™ Terminator Cycle

Sequencing Ready Reaction Kit with AmpliTaq®

DNA Polymerase, FS.

All other software requirements are unchanged; follow the user’s

manual for your ABI PRISM 373 DNA Sequencer model.

Sources The diskette supplied with the BigDye filter wheel includes the

mobility files needed to analyze data with the new sequencing

chemistries. These can also be obtained from the following sources:

♦Applied Biosystems site on the World Wide Web

(www.appliedbiosystems.com/techsupport)

♦Your local field applications specialist (call your local sales office

for more information)

User Bulletin: ABI PRISM® 373 DNA Sequencer Page 7 of 28

Installation of

Mobility Files

Perform the following steps to install the mobility files.

Software Settings To set up a run with the BigDye filter wheel, the following software

settings must be made.

♦Select filter set A.

♦Select the appropriate dye set/primer mobility file.

♦Select the dRhodamine instrument (matrix) file.

If you are running the BigDye filter wheel for the first time, refer to

“Creating a dRhodamine Instrument (Matrix) File” on page 8 and

“Installation of Mobility Files” above. To set up all other software

settings, refer to the user’s manual for your ABI PRISM 373 DNA

Sequencer model.

IMPORTANT When the BigDye filter wheel is installed, the instrument

will still indicate it is running filter set A with the old wavelengths. The kit

contains a label that lists the new wavelengths and indicates the instrument

has been upgraded to the BigDye filter wheel. At install, the service engineer

will place the label on the instrument.

Step Action

1In your Macintosh® system folder, locate the ABI folder.

2Install the following files in the ABI folder:

♦373BDP Rev

♦373BDP -21M13

♦373dRhod

♦373BDT

IMPORTANT Dye set/primer (mobility) file names for the

dRhodamine terminators are similar to those for the BigDye

terminators. Their respective mobility files can be mistaken for

each other easily without noticeably affecting the base spacing in

the data. In addition, if a mobility file for the wrong sequencing

chemistry is used, bases will be miscalled.

3Relaunch the Collection and/or Sequencing Analysis software if

either was open while the files were installed.

Note Sometimes it is necessary to restart the Macintosh

computer to use the dye set/primer files.

Page 8 of 28 User Bulletin: ABI PRISM® 373 DNA Sequencer

IMPORTANT When the BigDye filter wheel is installed, the FS rhodamine

terminators and FS dye primers will no longer be compatible. In addition, you

cannot perform GeneScan software analysis with the BigDye filter wheel

because the emissions from the GeneScan software dyes are not collected

efficiently.

Creating a dRhodamine Instrument (Matrix) File

Making a Matrix

from Matrix

Standards

Using the new dRhodamine sequencing chemistries requires a new

instrument (matrix) file. The new instrument file is made from the

matrix standards found in the ABI PRISM® dRhodamine Matrix

Standards Kit (P/N 403047). A set of four matrix standards only

needs to be run once to generate an instrument (matrix) file that is

used with all three of the dRhodamine-based chemistries.

An alternative method for preparing an instrument (matrix) file is

described in “Making a Matrix from a Sample File” on page 20.

The dRhodamine matrix standards are provided in a ready-to-use

format and are premixed with a blue dye for convenient gel loading.

Matrix standards are stable for six months at 2–6°C. The

dRhodamine matrix standards are listed below.

Table 3. dRhodamine Matrix Standards

Dye Tube Label

Color in Gel

Image for Filter

Set A

Color in

Electrophero-

gram

dichloro[R110] dR110 Matrix Std Blue Blue

dichloro[R6G] dR6G Matrix Std Green Green

dichloro[TAMRA] dTAMRA Matrix Std Yellow Black

dichloro[ROX] dROX Matrix Std Red Red

User Bulletin: ABI PRISM® 373 DNA Sequencer Page 9 of 28

Running Standards

To run matrix standards on the instrument:

Step Action

1Mix the matrix standards well.

2Prepare a separate loading cocktail as shown below for each of

the four matrix standards:

♦3

L of matrix standard

♦3

L of deionized formamide

! WARNING ! CHEMICAL HAZARD. Formamide is a

teratogen and is harmful by inhalation, skin contact, and

ingestion. Use in a well-ventilated area. Use

chemical-resistant gloves and safety glasses when handling.

! WARNING ! Please dispose of hazardous waste in

accordance with all local, state, and federal health and

environmental laws and regulations.

3Heat the cocktails at 95°C for 2 minutes. Place on ice until ready

to load.

IMPORTANT DNA samples should not be stored in

formamide for more than a few hours.

4Select filter set A.

5Perform the following steps to prepare the sample sheet:

a. List sample names exactly as they are written in the “Matrix

Standard” column of step 8 below.

b. Select any dye set/primer.

c. Select any instrument (matrix) file.

d. De-select Auto Analysis (refer to your user’s manual for

instructions).

Page 10 of 28 User Bulletin: ABI PRISM® 373 DNA Sequencer

6Load each of the four matrix standard cocktails into a separate

lane of the gels. Refer to the table below for the load amount that

corresponds to the comb you are using. It is a good idea to load

duplicates of each standard in case one of the lanes is unusable

due to an unforeseen gel artifact.

7Perform electrophoresis according to the user’s manual for your

instrument.

8After electrophoresis is complete, check the lane tracking in the

gel image for the matrix standards before making the matrix. The

matrix standards should display the following colors:

To run matrix standards on the instrument:

(continued)

Step Action

Instrument Loading Volume (

ABI 373A-18 comb 3.0

ABI 373A-24 comb 3.0

ABI 373A-36 comb 2.5–3.0

ABI 373AXL-48 comb 2.0

ABI 373AXL-64 comb 1.5

Matrix

Standard

Color in Gel

Image

Color in

Electropherogram

dR110 Blue Blue

dR6G Green Green

dTAMRA Yellow Black

dROX Red Red

User Bulletin: ABI PRISM® 373 DNA Sequencer Page 11 of 28

Making the Matrix

Put the correct data file for each matrix standard into the correct “box”

in the Data Utility application. You will be directed to do this at the

appropriate step.

IMPORTANT You need to make all three matrix files, even if you are only

using one dRhodamine-based chemistry. The Collection software will not run

with only a Taq terminator matrix in the file. An error message will appear

saying, “Multicomponent matrix error, Bad Data.” The T7 Terminator Matrix

file is needed to analyze BigDye terminator and dRhodamine terminator

chemistry data.

Table 4. Placement of Standards in the Data Utility Application

Box

Dye Primer

Matrix

Taq Terminator

Matrix

T7 Terminator

Matrix

C…dR110 dROX dR6G

A…dR6G dR6G dTAMRA

G…dTAMRA dR110 dROX

T…dROX dTAMRA dR110

To make the Dye Primer Matrix:

Step Action

1Launch the Data Utility software (located in the Utilities folder).

2From the Utilities menu, choose Make Matrix…

The Make Matrix dialog box appears as shown below. Verify that

the Dye Primer Matrix button at the lower left is selected.

Page 12 of 28 User Bulletin: ABI PRISM® 373 DNA Sequencer

3Click on the box for each nucleotide base and select the sample

file that corresponds to the correct matrix standard as shown in

the table below.

4For each matrix standard sample, start with the default value of

2000 for the start point. Start with the default value of 1500 for

the number of data points to analyze.

Note If the default values do not work, follow the instructions

for using other values in steps 8 and 9 below.

5Click New File…

A dialog window appears as shown below. Name the file dRhod

and save it in the ABI folder within the System folder.

To make the Dye Primer Matrix:

(continued)

Step Action

Box

Dye Primer

Matrix

C…dR110

A…dR6G

G…dTAMRA

T…dROX

User Bulletin: ABI PRISM® 373 DNA Sequencer Page 13 of 28

6The Make Matrix dialog box should look like that shown below.

a. Click OK.

The computer makes the matrix. When finished, a dialog window

appears with the message “Make matrix successfully

completed.”

b. Click OK.

7If the computer is unable to make a matrix, examine the raw data

again in the Sequencing Analysis software. If you used the

default values, then select new start points as directed in steps 8

and 9 below. If many peaks are off-scale, dilute the matrix

standards and rerun them.

To make the Dye Primer Matrix:

(continued)

Step Action

Page 14 of 28 User Bulletin: ABI PRISM® 373 DNA Sequencer

8If the matrix cannot be made with the default values, proceed

with steps a, b, and c below.

a. In the Sequencing Analysis software, open a matrix standard

sample and examine the raw data. An example is shown

below.

b. Select a starting point where there are no peaks and the

baseline is flat.

c. Select a number of data points to analyze such that no

peaks in the range are off-scale,

i.e.

, above 4000 relative

fluorescence units (RFU), and that the baseline at the end of

the range is flat. A typical number of data points is 1500.

9Repeat step 8 for each matrix standard sample. Record the

results for later use.

IMPORTANT The number of data points analyzed is the

same for each matrix standard. Choose starting points for each

sample such that all peaks are less than 4000 RFU and that both

the starting and ending points have flat baselines and no peaks.

To make the Dye Primer Matrix:

(continued)

Step Action

User Bulletin: ABI PRISM® 373 DNA Sequencer Page 15 of 28

To make the Taq Terminator Matrix:

Step Action

1In the Data Utility application, choose Make Matrix… from the

Utilities menu. The Make Matrix dialog box appears.

2In the Make Matrix dialog box, click the Taq Terminator Matrix

button at the lower left.

3Click on the box for each nucleotide base and enter the data file

that corresponds to the correct matrix standard as shown below.

IMPORTANT The order of matrix standard data files is

different from that in the Dye Primer Matrix (see Table 4 on

page 11).

4Enter the same numbers for each matrix standard sample in the

Start at and Points boxes as were used for the Dye Primer Matrix.

5Click Update File… A dialog window appears.

Box

Taq Terminator

Matrix

C…dROX

A…dR6G

G…dR110

T…dTAMRA

Page 16 of 28 User Bulletin: ABI PRISM® 373 DNA Sequencer

6Choose dRhod from the ABI folder within the System folder and

click Save. The Make Matrix dialog box is displayed.

Note The numbers in the Start at and Points boxes are default

values. Your numbers may vary.

7a. Click OK.

The computer makes the matrix. When finished, a dialog window

appears with the message “Make matrix successfully completed.”

b. Click OK.

To make the Taq Terminator Matrix:

(continued)

Step Action

User Bulletin: ABI PRISM® 373 DNA Sequencer Page 17 of 28

To make the T7 Terminator Matrix:

Step Action

1In the Data Utility application, choose Make Matrix… from the

Utilities menu. The Make Matrix dialog box appears.

2In the Make Matrix dialog box, click the T7 Terminator Matrix

button at the lower left.

3Click on the box for each nucleotide base and enter the data file

that corresponds to the correct matrix standard as shown in the

table below (note the order of the matrix standard files).

4Enter the same numbers for each matrix standard sample in the

Start at and Points boxes as were used in the Dye Primer Matrix

and Taq Terminator Matrix.

5Click Update File… A dialog window appears.

Box

T7 Terminator

Matrix

C…dR6G

A…dTAMRA

G…dROX

T…dR110

Page 18 of 28 User Bulletin: ABI PRISM® 373 DNA Sequencer

6Choose dRhod from the ABI folder within the System folder and

click Save.

The Make Matrix dialog box should look like that shown below.

Note The numbers in the Start at and Points boxes are default

values. Your numbers may vary.

7a. Click OK.

The computer makes the matrix. When finished, a dialog window

appears with the message “Make matrix successfully completed.”

Click OK.

To make the T7 Terminator Matrix:

(continued)

Step Action

User Bulletin: ABI PRISM® 373 DNA Sequencer Page 19 of 28

To check the instrument file:

Step Action

1From the Utilities menu, choose Copy Matrix…

2Under Source, select Instrument file and choose dRhod from the

ABI folder within the System folder.

The three matrix files within the dRhod instrument file appear as

shown below.

3Make sure that all three matrix files have numbers that range from

0–1. The numbers on the diagonals from top left to bottom right

should be 1. If not, then repeat the matrix-making procedure

starting with “To make the Dye Primer Matrix:” on page 11.

Note The corresponding numbers for all three matrix files will

be the same.

4Click Cancel.

5Open or restart the Sequencing Analysis software and use

dRhod as the instrument file to analyze your sequencing data.

Page 20 of 28 User Bulletin: ABI PRISM® 373 DNA Sequencer

Making a Matrix

from a Sample File

An instrument file can be made from matrix standards as explained

above, or it can be made from a sample file that was run with one of

the three dRhodamine-based chemistries. This procedure requires

fewer steps than running matrix standards, however, the matrix made

from a sample file may not be as good as one made from matrix

standards. The quality of a matrix file made from a sample file

depends on the quality of the sample file used.

The best samples to choose for making a matrix have approximately

25% each of A, C, G, and T. A good example of this is the pGEM DNA

with the -21M13 primer that is included as a control in every Ready

Reaction Sequencing Kit. To create a matrix from a sample file, follow

the steps below.

To create a matrix from a sample file:

Step Action

1Prepare a sequencing reaction according to the protocol booklet

for the chemistry you choose.

2Select filter set A.

3Perform the following steps to prepare the sample sheet:

a. List sample names.

b. Select any dye set/primer.

c. Select any instrument (matrix) file.

d. De-select Auto Analysis (refer to your user’s manual for

instructions).

4Load the sample according to the load chart for your comb size.

See Table 5 on page 23.

5Perform electrophoresis according to the user’s manual for your

instrument.

6Before making the matrix, verify that lane tracking is accurate.

Adjust if necessary.

7Duplicate the unanalyzed sample file three times. Use the

Duplicate command from the File menu in the Finder. You will

have a total of four copies of the same sample file with the

following names:

♦Sample name

♦Sample name Copy 1

♦Sample name Copy 2

♦Sample name Copy 3

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Thermo Fisher Scientific Using the ABI 373 BigDye Filter Wheel: ABI PRISM® 373 DNA Sequencer Owner's manual

Thermo Fisher Scientific Using the ABI 373 BigDye Filter Wheel: ABI PRISM® 373 DNA Sequencer Owner's manual

Thermo Fisher Scientific Using the ABI 373 BigDye Filter Wheel: ABI PRISM® 373 DNA Sequencer Owner's manual

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