A Samplesheet program originally was written in Think C on the Macintosh by LaDeana Hillier at the Washington University Genome Center for the ABI 373 and later modified for use at OU by Bruce Roe. Subsequently, Bruce Roe and Steve Kenton created a version of the Sample Sheet program for the 377 called, "Make ABI Sample Sheet", based on the naming convention guidelines listed below.
After opening the program by double clicking on the icon, the user is prompted for answers to the various questions required to determine the correct block, sample name, project code, loading device, and primer designation. The program then automatically creates a "Sample Sheet" in the ABI Samplesheet folder specific for that run.
The main advantage of the "Make ABI sample sheet" program is that it can create Samplesheets for multiple gel loading formats:
The first format is used when samples are loaded one at a time either with the Eppendorf pipet with duck bill tip or the 1cc glass Hamilton syringe. This loading format requires a list in sequential order from, a01-d12, for 48 samples and the loading procedure is as follows:
Load the odd numbered samples, i.e. every other lane beginning with lane 1, electrophorese the samples on for 5 minutes, and then load the even numbered samples to the even numbered lanes, and begin the actual run.
The second format is used in conjunction with an eight sample 1cc glass Hamilton multichannel syring #84503 and has options for loading 2 ABI 373/377s using the 48 sample lane comb and samples from 1 96-well microtiter plate and for loading 3 ABI 377s with the XL upgrade using the 64 sample lane comb and 2 96-well microtiter plates.
One 96-well microtiter plate (96 samples) are loaded onto 2X48-lane ABI 373/377s with or without the XL upgrade.
Because the samples in the microtiter plate are stagger loaded onto 2 ABI 373/377s in sets of 8, the loading and samplesheet are rather complex. By following the protocol below, the "SampleSheet" generated will correspond correctly to the original samples in the microtiter plate.
Load rows 1 and 2 of the microtiter plate to lanes 1,4,7, etc and 25,28,31, etc on the first ABI and run the electrophoresis for 5 minutes while loading samples in rows 7 and 8 in the microtiter plate to lanes 1,4,7, etc and 25,28,31, etc on the second ABI and run these on for 5 minutes. Then go back to the first ABI and load the samples in rows 3 and 4 to lanes 2,5,8, etc and lanes 26,29,32, etc on the first ABI, rows 9 and 10 of the microtiter plate to lanes 2,5,8, etc and lanes 26,29,32, etc on the second ABI with another 5 minute electrophoresis after each ABI loading. Repeat this one more time to complete loading both ABI's, with the 5 minute electrophoresis after each loading. Then, begin the runs.
Two 2X96-well microtiter plates (192 samples) to be loaded onto 3X64-lane ABI 377s with the XL upgrade.
Because the samples in the two microtiter plates are stagger loaded onto 3 ABI 377s in sets of 8, the loading and samplesheet are rather complex. By following the protocol below, the "SampleSheet" generated will correspond correctly to the original samples in the microtiter plates.
Load rows 1 and 2 of the first microtiter plate to lanes 1,5,9, etc and 33,37,41, etc on the first ABI and run the electrophoresis for 5 minutes while loading samples in rows 9 and 10 in the microtiter plate to lanes 1,5,9, etc and 33,37,41, etc on the second ABI and run these on for 5 minutes while loading samples in rows 5 and 6 in the second microtiter plate to lanes 1,5,9, etc and lanes 33,37,41, etc on the third ABI and run those on for 5 minutes. Then go back to the first ABI and load the samples in rows 3 and 4 to lanes 2,6,10, etc and lanes 34,38,42, etc on the first ABI, rows 11 and 12 of the first microtiter plate to lanes 2,6,10, etc lanes 34,38,42, etc on the second ABI and and rows 7 and 8 on the second microtiter plate to lanes 2,6,10, etc and lanes 34,38,42, etc on the third ABI with another 5 minute electrophoresis after each ABI loading. Repeat this two more times to complete loading all 3 ABI's with the reactions from both microtiter plates, with again the 5 minute electrophoresis after each loading. Then, begin the runs.
Although it is not available yet from ABI, a 96 lane samplesheet option is provided in anticipation of eventual upgrades to the 377 hardare and software. Being table driven, the software source code can be easily modified for any number of lanes in any loading order desired.
This directory contains 4 files in addition to this readme file: 373_program_sit.hqx 377_program_sit.hqx 373_source_sit.hqx 377_source_sit.hqx
The files 373_program_sit.hqx and 377_program_sit.hqx contain the Macintosh executable files needed to run the SheetWriter program. The 373 version of SheetWriter runs on the non-PowerMac 68xxx Macintosh connected to the 373, and the 377 version of Sheetwriter runs on the PowerMac attached to a 377.
The files 373_source_sit.hqx and 377_source_sit.hqx contain the ThinkC Symantec version 7.0 source code for the 373 and 377 versions of SheetWriter.
These files can be downloaded with Netscape and will automatically be "un-bin-hexed" and "un-stuffed" if yoou have Stuffit Expander installed and recognized by Netscape.
Alternatively, these files can be transfered by ftp in ASCII mode and then can be "un-bin-hexed" using Stuffit1.5.1 or other programs that "decode binhex files". Then, you should use Stuffit1.5.1 or Stuffit Expander to uncompress the Stuffit archive.
I used Stuffit1.5.1 to create the Stuffit archives (xxx.sit files) and the BinHex files (xxx_sit.hqx).
The 377 version allows various loading options, including individual sample loadings (typically with the Eppendorf pipetman and duck-bill tips), as well as multiple sample loadings (with the Hamilton 8 sample syringe with 0.9mm needle spacing and the 0.2mm OD needles). The latter is useful when loading directly from a microtiter plate and the 377 version of SheetWriter, writes the samplesheet taking into account the staggered loading order.
We have found that a 5 minute electrophoresis is optimal between sample loadings. A 3 minutes or less electrophoresis is non-ideal and >6 minutes is unnecessary.
The guidelines for the naming convention used are as follows:
1. The sample name must be a total of 10 characters. For large insert shotgun clone libraries, i.e. BACs, Fosmids, etc, use: (a-z)(1-9)(a-z)(01-99)(a-z)(a-z).(f,r,s)(1-9) or (a-z)(1-9)(a-z)(01-99)(a-z)(1-9).(f,r,s)(1-9) Example: a1b01cc.f1 or a1b01d3.f1 For EST and WGS library sequencing, use: (0-9)(1-9)(a-z)(01-99)(a-z)(a-z).(f,r,s)(1-9) or (0-9)(1-9)(a-z)(01-99)(a-z)(1-9).(f,r,s)(1-9) Example: 01b01cc.f1 or 21b01d3.f1 For test runs, use: t(1-9)(a-z)(01-99)(a-z)(a-z).(f,r,s)(1-9) Example: t1b01cc.f1 or t9b01d3.f1 2. The first two characters are to designate the template block. Where the first character is a letter, a-z (or number 0-9), followed by a single digit number, i.e. 1-9. Example: a1 (or 01 or 21) 3. The next three characters are to designate the individual well in the template block. Where the first character is a letter, a-h, followed by a double digit number, 01-12. Example: a01 4. The next two characters designate the project code with each individual project being assigned a specific code. This code consists of either two letters, a-z, or one letter followed by a number, 1-9. Example: aa or a1 5. It also is necessary to identify the primer used for sequencing and whether the clone is being resequenced for gap closure. The primer used is designated by, .f (forward), .r (reverse) or .s (synthesized primer) and each time the clone is sequenced with a given primer, it is assigned a number (1-9) where 1 represents the shotgun phase and 2-9 represents resequencing during closure. Example: .f1 or .r1 or .s1 (note that the "." is mandidatory and added by the program automatically)
Bruce Roe, email@example.com