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Assessment of Clone Identity and Sequence Fidelity
Within a Commercially Distributed Subset of IMAGE Clones
R.G Halgren, M.R. Fielden, C.J. Fong, T.R. Zacharewski
Abstract
This report documents the error rate in a commercially distributed subset of the IMAGE Consortium mouse cDNA clone collection. After isolation of plasmid DNA from 1189 bacterial stock cultures, only 62.2 percent were uncontaminated and contained cDNA inserts that had significant sequence identity to published data for the ordered clones. An agarose gel electrophoresis prescreening strategy identified 361 stock cultures that appeared to contain two or more plasmid species. Isolation of individual colonies from these stocks demonstrated that 7.1 percent of the original 1189 stocks contained both a correct and an incorrect plasmid. 5.9 percent of the original 1189 stocks contained multiple, distinct, incorrect plasmids, indicating the likelihood of multiple contaminating events. While only 739 of the stocks purchased contained the desired cDNA clone, agarose gel prescreening, colony isolation, and similarity searching of dbEST allowed for the identification of an additional 420 clones that would have otherwise been discarded. Considering the high error rate in this subset of the IMAGE cDNA clone set, the use of sequence verified clones for cDNA microarray construction is warranted.
Supplementary Data
| Description |
Notes |
Available Files |
| Summary
of results by stock ordered |
This file presents the information for the clone order submitted to Research Genetics, including the IMAGE clone ID ordered, the library from which the clone is derived, location in IMAGE stock plates, and location on the stock plates we received. Also included is an analysis of which stocks were correct, incorrect, contaminated, or failed to grow. |
per_stock.xls |
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| Sequence
Verification Data Summary |
Excel file with four summary pages.
1 - Correct
sequences from prescreened clones (primary)
2 - Incorrect sequences
from prescreened clones with BLAST data for clone identification
3-
Correct sequences from Isolated clones (secondary)
4- Incorrect
sequences from isolated clones with BLAST data for clone identification |
webdata_totals.xls |
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| BLAST Data
(Isolated Clones) |
The results of attempts to identify unknown sequences
obtained during physical isolation of clones. Up to 10 significant hits
per query (significance defined as E < 1e-50) are examined against
the UniGene database to obtain a UniGene cluster ID. Note that not all
ESTs are found in a UniGene cluster. |
blast_negatives1.htm |
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| Comparison of Negative Clones |
A pairwise comparison of all clones derived from a
single stock well using the BLAST 2 Sequences tool. Note that short
matches in the 5' region of a sequence were not considered to be
evidence of identity. This is probably cloning vector sequence. |
compare_negatives.htm |
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