Batch 5, which includes the bra clasp (Rep. 165), shows very serious problems. Demonstrable machine malfunction and obvious contamination render unreliable all of the quantification results for Batch 5; in other words, there is no reliable quantification result to show “abundant” DNA in Rep. 165b, as has been claimed. In addition, Rep. 165b (bra clasp hooks) apparently was profiled twice, with the results of the first analysis being suppressed by the prosecution. Record-keeping anomalies, together with the belated re-run of 165b, even suggest tampering with the results of the analyses for this exhibit. Generally, the contamination, machine malfunction, processing irregularities and record-keeping issues render unreliable the results reported for Batch 5, and raise serious questions about the integrity of the lab’s work.
This batch consists of evidence collected from the cottage during the “second” visit on December 18, 2007. The extractions were run on December 29, 2007, quantifications were run on January 3, 2008, and we can estimate that the main sequencing occurred between January 3-4, with partial plates likely being processed the following week, January 7- 9. The results of the bra clasp testing were leaked to the press on or about January 10, 2008.
The Batch 5 profiles were generated from one main plate, 410, followed by several apparently partial plates, 413 (autosomal and Y), 414 (bra clasp hooks only) and 417 (one Y only). The main plate, no. 410, consists of 32 profiles, 17 of which are suppressed, for a suppression rate of 53% (40% excluding luminol and unknown samples). The suppressed profiles come from: luminol samples (6), items from Knox’s room (3), Kercher’s effects (3), a mop, a negative control, and unknown samples (3). Curiously, four of the missing profiles (ID Nos. 681, 700, 702 and 703), including, notably, Rep. 165b (i.e., the bra clasp hooks), appear to have been run subsequently, in plate nos. 413 and 414. It is unknown whether the defense was advised of the re-runs, and Prof. Potenza’s known reports do not cover any of the testing in this batch.
The quantification records for this batch demonstrate contamination. The lab used a dilution standard of known concentration—23 pg/µL—as the positive control for the quantification run (see RT-qPCR Run No. 570 at wells H11-H12). This positive control, however, did not quantify at 23 pg/µL as should be expected, but rather, at 108 pg/µL (average of quantification values shown in RT-qPCR Run No. 570 at wells H11-H12). In other words, the positive control contained almost five times as much DNA as it should have.
This demonstrates that a substantial amount of DNA was introduced into this control via a contamination event.
3. Machine Malfunction
Not only is contamination in the Real Time run for Batch 5, but it is also evident that the machine was malfunctioning, likely due to a failure to maintain it properly. The dilution standards (i.e., dilutions used to calibrate the machine readings) for the quantification run are shown at wells A1 through B9, and when the Ct results for these standards are compared to other runs, it can be seen that the dilution standards for RT-qPCR Run 570 are approximately 4 Ct higher than for most of the other runs (there are a few prior RT-qPCR Runs for which the machine also appears to be malfunctioning). This discrepancy demonstrates that there is a problem with the operation of the machine. Notably, Batch 5 was the last batch for which the lab used the ABI 7700 Real Time machine: all subsequent samples were quantified via a Fast Time 7500 machine, demonstrating that the lab was aware of, and reacted to, the malfunctioning real Time machine.
4. Transposition of Sub-Rep Designations
There is an abnormality in the identification of Rep. 165’s sub-Reps, which suggests error or even the possibility of tampering. Rep. 165 yielded two traces: No. 48896 (hooks) and 48897 (fabric). There is a transposition in the designation of sub-Reps, however: the first in sequence (48896) is designated as sub-rep. “b,” and the later in sequence (48897) is designated as sub–rep. “a.” In prior samples, sub-rep numbering always follows sequentially from the trace numbering, e.g., 47329=36a, 47330=36b, 47331=36c, etc. The apparent transposing of Rep. 165’s sub-rep. designations, so that sub-rep “b” precedes sub-rep. “a” is unprecedented among the approximately 100 other samples in this case that involve sub-reps and sub-rep numbering.
5. Apparent Re-Run of 165b
Reconstruction of plate 410 demonstrates that Rep 165b’s profile was run once, suppressed, and re-run in a separate plate at the very end of the batch. It is only this second run of 165b that has been produced by the prosecution and touted as an incriminating profile, with the first run remaining unacknowledged and indeed secreted by the prosecution.
As can be seen in the chart for Batch 5, the amplification corresponding to Rep 165b is ID No. 681. This ID No. falls within the range of ID Nos. contained within the 32-sample plate no. 410, which spans ID Nos. 680 through 711. Yet, the prosecution has never produced an electropherogram corresponding to plate no. 410, ID 681. Instead, the prosecution has produced an ID No. 681 purportedly generated in plate no. 414. This electropherogram for plate no. 414, ID 681, is remarkable, because it is the only known electropherogram produced for this plate (a single-sample plate being a phenomena that is seen in only one other example in this case). In addition, plate no. 414 itself is remarkable, because it sequentially follows plate no. 413, which contains Y profiles and thus should mark the end of batch no. 5. In effect, the electropherogram that has been produced for Rep. 165b is from the wrong plate and appears exceptionally to have been generated after the end of the relevant batch analyses.
The inconsistency in the plate number for ID 681, and the fact that there is only a solitary electropherogram from plate no. 414, leads to the conclusion that there is an undisclosed profile corresponding to plate 410, ID 681, and that plate 414, ID 681 is a re-run of this prior sequencing process. Taken together with the transposition of the Sub-Rep designations for 165b, and the occurrence of contamination within this batch, it can be seen that there is substantial irregularity and perhaps tampering that has occurred concerning 165b (bra clasp hooks).