Artifacts Identified Post-Developmental Validation: AmpFLSTR Identifiler Plus PCR Amplification Kit

Transcription

1 March 29, 2018 TECHNICAL NOTE Artifacts Identified Post-Developmental Validation: AmpFLSTR Identifiler Plus PCR Amplification Kit The purpose of this document is to assist with data interpretation by providing a repository of artifacts, identified and characterized post-developmental validation of the Identifiler Plus kit (PNs: , , A26182 and A26364) as a result of investigating customer reports. Many of the artifacts included in this document fall below the peak amplitude threshold (PAT) used during developmental validation or are attributable to specific sample types that were not encountered during developmental validation. The Identifiler Plus kit shares primer sequences with the AmpFLSTR Identifiler PCR Amplification Kit and AmpFLSTR Identifiler Direct PCR Amplification Kit. Therefore, the artifacts described in this technical note may also be observed in the Identifiler and Identifiler Direct kits. The three kits differ in master mix formulation and primer concentrations, which may impact artifact signal intensities and ratios to parent peaks. Background During developmental validation of the Identifiler Plus PCR Amplification Kit, a 50 RFU PAT was applied to data generated on the 3130xL Genetic Analyzer. Many laboratories, following their internal Identifiler Plus validations, implemented PATs below 50 RFU and/or have transitioned to the 3500/xL Genetic Analyzer that employs a broader dynamic range and cleaner baseline than preceding capillary electrophoresis platforms. As a result of lower analysis thresholds and increased sensitivity and cleaner baselines, low level artifacts not identified during developmental validation have been reported. Due to the wide variety of sample and substrate types processed in forensic laboratories, it is not feasible to process all sample and substrate type combinations during STR kit development and validation testing. Instead, representative samples and substrates are selected and analyzed during this process. Therefore, it is possible for sample-specific artifacts to be identified through customer reports and subsequently tested and characterized in the Thermo Fisher Scientific HID Laboratory. Such artifacts are included in this document to assist with interpretation of data generated from similar sample or substrate types. A variety of mechanisms, such as dye byproducts, the formation of secondary structures, nontraditional stutter, non-specific binding, or non-human interaction can introduce artifacts into PCR STR data. The detectable presence and intensity (peak height) of these types of artifacts can be 1

2 dependent upon amplification and/or electrophoresis conditions, such as elevated PCR cycle numbers or CE system sensitivity differences. Method Artifacts included in this technical note were detected and characterized post-product development. Artifacts that are the result of standard PCR phenomena, such as traditional stutter or minus A peaks, are not included. See the Identifiler Plus kit user guide (publication number ) for information on these types of artifacts. Although the Identifiler Plus kit developmental validation occurred on a 3130xL genetic analyzer, the data herein is specific to Identifiler Plus kit data run on a 3500 series instrument, and unless otherwise noted, was generated following all manufacturer recommended PCR and electrophoresis conditions, as documented in the Identifiler Plus kit user guide and the 3500/3500xL HID user bulletin (part number ). The artifacts summarized in this document may appear on other capillary electrophoresis platforms, although peak heights and base pair sizes may differ. Should new artifacts be identified and characterized, they will be periodically added to this document. Summary Table: Click on the hyperlinks for more information. Dye Channel Locus Artifact ID Approximate Base Typical Allele Pair (bp) Size Call N/A IDP_FAM bp OMR or OL D8S1179 IDP_D bp OL 6-FAM IDP_D bp OL or 28.2 D21S11 IDP_D bp OL or 38 TH01 IDP_TH01-1 N-8 to N-12 bp Varies VIC D13S317 IDP_D bp OL D16S539 IDP_D bp OL or 7 D19S433 IDP_D bp OL IDP_vWA bp OL IDP_vWA bp OL or 12 vwa NED IDP_vWA bp OL or 24 IDP_vWA bp OL or 24.1 TPOX IDP_TPOX-1 N-14 to N-15 bp Varies IDP_TPOX-2 N+3 bp OL PET N/A IDP_PET bp N/A 2

3 Detailed Artifact Descriptions Dye Channel: 6-FAM Locus: N/A Artifact ID: IDP_FAM-1 Location: ~ bp o Related to the IDP_vWA-1 artifact also listed in this document. o Observed in a swab of a cooked pork rib o Sequencing studies have shown that the IDP_FAM-1 artifact sequence has no significant homology to the human genome, and at the time of the NCBI BLAST search there were no significant matches to genetic sequences in the database.** o For comparison purposes, a cooked pork rib was purchased from a Chinese restaurant and used as a control. The meat and bone of the pork rib was swabbed. DNA from the swab was isolated, quantitated, amplified with the Identifiler Plus kit, and injected on a 3500xL Genetic Analyzer. The control DNA profile from the cooked pork rib, shown in Figure 2, displayed the reported artifact peaks IDP_FAM- 1 and IDP_vWA-1. Figure 1. Sample dependent artifact (IDP_FAM-1), obtained from an evidence swab of a pork rib, located at bp in the FAM dye channel, between the D21S11 and D7S820 markers. The y-axis is scaled to 1000 RFU, and the artifact is 966 RFU. 3

4 Figure 2. Artifacts generated from 8 ng of DNA isolated from a control swab of a cooked pork rib. The two peaks at bp in D7S820 (red circle) and bp in vwa (green circle) are consistent with the IDP_FAM-1 and IDP_vWA-1 artifacts described in this document. The y-axis is scaled to 200 RFU, and the artifacts are 29 RFU and 27 RFU respectively. The PET artifact at bp, and associated pullup boxed in blue, is consistent with the IDP_PET-1 artifact. Locus: D8S1179 Artifact ID: IDP_D8-1 Location: ~ bp o Related to the IDP_vWA-2 artifact also listed in this document. o Observed in a swab of a partially eaten burrito o Sequencing studies have shown that the D8S1179 artifact sequence has partial homology with the Triticum aestivum (common wheat) genome. At the time of the search, the NCBI BLAST statistics for this match included an E value of 4.0e -67 with 97% query coverage and 94% identity.** o For confirmation purposes, wheat germ was purchased from a supermarket. DNA from the wheat germ was isolated, amplified with the Identifiler Plus kit, and injected on a 3500xL Genetic Analyzer. The wheat germ DNA profile, shown in Figure 4, displayed the reported artifact peaks IDP_D8-1 and IDP_vWA-2. 4

5 Figure 3. Sample dependent artifact (IDP_D8-1), obtained from a swab of a burrito, at bp in D8S1179. The y-axis is scaled to 1000 RFU, and the artifact is 387 RFU. Figure 4. Identifiler Plus electropherogram generated from the DNA extraction, amplification and 3500xL fragment analysis of store purchased wheat germ. The two peaks at bp in D8S1179 and bp in vwa are consistent with the IDP_D8-1 and IDP_vWA-2 artifacts described in this document. The y-axis is scaled to 130 RFU, and the artifacts are 111 RFU and 110 RFU respectively. Locus: D21S11 Artifact ID: IDP_D21-1 Location: ~201.5 bp o Related to the IDP_D13-1 and IDP_D19-1 artifacts also listed in this document. 5

6 o o o The customer report of this artifact indicated that a police canine retrieved the item of evidence tested. Internal investigation for this artifact included controls consisting of reference buccal samples from three different domesticated dogs. One of the three canine controls produced all three reported artifacts (IDP_D21-1, IDP_D13-1 and IDP_D19-1), while the other two reference dog samples did not produce any of the reported artifacts. Sequencing studies have shown that the D21S11 artifact sequence has partial homology with a variety of different mammals, including canine. Figure 5. Sample dependent artifact (IDP_D21-1), obtained from a reference buccal swab collected from a domesticated dog, at bp in D21S11. Additional PCR cycles beyond the standard 28 cycles were utilized to better isolate the artifact. The y-axis is scaled to 3000 RFU, and the artifact is 190 RFU. The human STR profile is a mixture, likely from the dog-owner s family. Artifact ID: IDP_D21-2 Location: ~240 bp o Has been observed in sample types potentially exposed to fecal matter, for example rectal and anal swabs. o Sequencing studies have shown that the artifact sequence has partial homology with the Porphyromonas asaccharolytica (gram-negative anaerobic bacteria) genome. At the time of the search, the NCBI BLAST statistics for this match included an E value of 2.0e -53 with 96% query coverage and 81% identity.** o This artifact has also been observed in the GlobalFiler kit at ~250 bp in the TAZ dye channel (GF_TAZ-1). 6

7 Figure 6. Sample dependent artifact (IDP_D21-2), obtained from an anal swab, at bp in D21S11. The y-axis is scaled to 3000 RFU, and the artifact is 1066 RFU. Dye Channel: VIC Locus: TH01 Artifact ID: IDP_TH01-1 Location: N-8 to N-12 bp (typically observed ~8-12 nucleotides before the parent TH01 peak(s)) Probable cause: DNA template dependent artifact likely caused by formation of a secondary structure in the target sequence Typical signal intensity: relative to DNA input amount (~0.4% to 1.1% of the parent peak height)* o This artifact has also been observed in the GlobalFiler PCR Amplification Kit at 10 to 12 nucleotides before the parent TH01 peak(s) (GF_TH01). Figure 7. PCR artifact (IDP_TH01-1), obtained from 1 ng 9947A control DNA, located 8.06 bp before the 8 allele in TH01. The y-axis is scaled to 200 RFU and the artifact is 28 RFU, which is 1.1% of the parent 8 allele. Locus: D13S317 Artifact ID: IDP_D13-1 Location: ~204 bp 7

8 o Related to the IDP_D21-1 and IDP_D19-1 artifacts also listed in this document. o The customer report of this artifact indicated that a police canine retrieved the item of evidence tested. o Internal investigation for this artifact included controls consisting of reference buccal samples from three different domesticated dogs. One of the three canine controls produced all three reported artifacts (IDP_D21-1, IDP_D13-1 and IDP_D19-1), while the other two reference dog samples did not produce any of the reported artifacts. o Sequencing studies have shown that the D13S317 artifact sequence has partial homology with the Canis Familiaris chromosome 16 genome. At the time of the search, the NCBI BLAST statistics for this match included an E value of 1.0e -88 with 96% query coverage and 99% identity.** Figure 8. Sample dependent artifact (IDP_D13-1), obtained from a reference buccal swab collected from a domesticated dog, at bp in D13S317. Additional PCR cycles beyond the standard 28 cycles were utilized to better isolate the artifact. The y-axis is scaled to 3000 RFU, and the artifact is 493 RFU. The human STR profile is a mixture, likely from the dog-owner s family. Locus: D16S539 Artifact ID: IDP_D16-1 Location: ~ bp o Has been observed in a cigarette butt and underwear o Sequencing studies have shown that the artifact sequence has partial homology with the Stenotrophomonas maltophilia genome. At the time of the search, the NCBI BLAST statistics for this match included an E value of 1.0e -90 with 96% query coverage and 90% identity.** 8

9 Figure 9. Sample dependent artifact (IDP_D16-1), obtained from a cigarette butt, at bp in D16S539. The y-axis is scaled to 650 RFU, and the artifact is 117 RFU. Dye Channel: NED Locus: D19S433 Artifact ID: IDP_D19-1 Location: ~139 bp o Related to the IDP_D21-1 and IDP_D13-1 artifacts also listed in this document. o The customer report of this artifact indicated that a police canine retrieved the item of evidence tested. o Internal investigation for this artifact included controls consisting of reference buccal samples from three different domesticated dogs. One of the three canine controls produced all three reported artifacts (IDP_D21-1, IDP_D13-1 and IDP_D19-1), while the other two reference dog samples did not produce any of the reported artifacts. o Sequencing studies have shown that the D19S433 artifact sequence has no significant homology to human or canine DNA but has partial homology to a fungal species known to infect the skin of domesticated dogs and cats. 9

10 Figure 10. Sample dependent artifact (IDP_D19-1), obtained from a reference buccal swab collected from a domesticated dog, at bp in D19S433. Additional PCR cycles beyond the standard 28 cycles were utilized to better isolate the artifact. The y-axis is scaled to 3000 RFU, and the artifact is 1101 RFU. The human STR profile is a mixture, likely from the dog-owner s family. Locus: vwa Artifact ID: IDP_vWA-1 Location: ~ bp o Related to the IDP_FAM-1 artifact also listed in this document. o Observed in a swab of a cooked pork rib o Sequencing studies have shown that the IDP_vWA-1 artifact sequence has no significant homology to the human genome, and at the time of the NCBI BLAST search there were no significant matches to genetic sequences in the database.** o For comparison purposes, a cooked pork rib was purchased from a Chinese restaurant and used as a control. The meat and bone of the pork rib was swabbed. DNA from the swab was isolated, quantitated, amplified with the Identifiler Plus kit, and injected on a 3500xL Genetic Analyzer. The control DNA profile from the cooked pork rib, shown in Figure 2, displayed the reported artifact peaks IDP_FAM- 1 and IDP_vWA-1. 10

11 Figure 11. Sample dependent artifact (IDP_vWA-1), obtained from an evidence swab of a pork rib, located at bp in the vwa locus. The y-axis is scaled to 550 RFU, and the artifact is 342 RFU. Artifact ID: IDP_vWA-2 Location: ~ bp o Related to the IDP_D8-1 artifact also listed in this document. o Observed in a swab of a partially eaten burrito o Sequencing studies have shown that the vwa artifact sequence has partial homology with the Triticum aestivum (common wheat) genome. At the time of the search, the NCBI BLAST statistics for this match included an E value of 1.0e -34 with 84% query coverage and 90% identity.** o For confirmation purposes, wheat germ was purchased from a supermarket. DNA from the wheat germ was isolated, amplified with the Identifiler Plus kit, and injected on a 3500xL Genetic Analyzer. The wheat germ DNA profile, shown in Figure 4, displays the reported artifact peaks IDP_D8-1 and IDP_vWA-2. Figure 12. Sample dependent artifact (IDP_vWA-2), obtained from a swab of a burrito, at bp in vwa. The y-axis is scaled to 1000 RFU, and the artifact is 467 RFU. 11

12 Artifact ID: IDP_vWA-3 Location: ~206 bp o Has been observed in a variety of crime scene related sample types o Sequencing studies have shown that the artifact sequence has partial homology to a variety of different bacterial species commonly found in soil, with no sequence homology to the human genome. Figure 13. Sample dependent artifact (IDP_vWA-3), obtained from a touch swab of an unidentified evidence item, at bp in vwa. The y-axis is scaled to 1000 RFU, and the artifact is 967 RFU. Artifact ID: IDP_vWA-4 Location: ~207 bp o Has been observed in a variety of crime scene related sample types typically exposed to the outdoor environment. o Sequencing studies have shown that the vwa artifact sequence has partial homology to yeast and fungal species commonly found in the environment, such as from plants, soil and decaying organic matter, with no sequence homology to the human genome. o This artifact has also been observed in the GlobalFiler kit at ~207 bp in the D21S11 locus (GF_D21-1). 12

13 Figure 14. Sample dependent artifact (IDP_vWA-4), obtained from an unidentified evidence sample, at ~207 bp in vwa. Additional PCR cycles beyond the standard 28 cycles were utilized to better isolate the artifact. The y-axis is scaled to 10,000 RFU and the artifact is ~7,000 RFU. Locus: TPOX Artifact ID: IDP_TPOX-1 Location: N-14 to N-15 bp (typically observed ~14-15 nucleotides before the parent TPOX peak(s)) Probable cause: DNA template dependent artifact likely caused by formation of a secondary structure in the target sequence Typical signal intensity: relative to DNA input amount (~0.4% to 1% of the parent peak height)* o This artifact has also been observed in the GlobalFiler kit at ~24 nucleotides before the parent TPOX peak(s) (GF_TPOX-1). Figure 15. PCR artifact (IDP_TPOX-1), obtained from 1 ng 9947A control DNA amplified at 30 cycles, located at bp before the 8 allele in TPOX. The y-axis is scaled to 1000 RFU and the artifact is 387 RFU, which is 1% of the parent 8 allele. Artifact ID: IDP_TPOX-2 Location: N+3 bp (typically observed ~3 nucleotides after the parent TPOX peak(s)) Probable cause: Post-PCR artifact caused by binding of a GeneScan 600 LIZ Size Standard manufacturing byproduct to a complementary sequence in TPOX Typical signal intensity: varies relative to multiple variables, such as: o An increase in intensity with time after denaturation 13

14 o A decrease in intensity on the 3500 platform due to improved temperature control o Reports/observations of this artifact are rare o Only the TPOX locus is affected o Can affect allelic ladder and amplified samples o The artifact may occur when amplicon containing TPOX is co-injected with GeneScan600 LIZ Size Standards (part numbers or ) and when conditions are favorable for promoting artifact formation, such as: >24 hours between CE sample-prep and CE injection Failure to use Hi-Di Formamide or use of expired or compromised Hi-Di Formamide Failure to heat denature and snap-cool CE-prepared samples prior to injection o The artifact is not observed with GeneScan 500 LIZ Size Standard (part number ) o Follow all CE sample-prep guidelines and use fresh CE preparations to avoid formation of the artifact at detectable levels Figure 16. Post-PCR artifact (IDP_TPOX-2), increasing in signal intensity with increasing time following CE prep and initial heat denaturation. Re-denaturation via a second heat step prior to reinjection reduces the artifact but does not eliminate it. Y-axis is scaled to 700 RFU and the artifact ranges from ~50 RFU to ~650 RFU over the span of 49 hours. Dye Channel: PET Locus: N/A Artifact ID: IDP_PET-1 14

15 Location: ~101 bp o Pig bone was purchased from a supermarket and marrow was recovered from the bone. DNA from the pig bone marrow was isolated, quantitated, amplified with the Identifiler Plus kit, and then injected on a 3500xL Genetic Analyzer. o As per the Species Specificity section of the Identifiler Plus user guide, several animal species (dog, horse, sheep and cow) have produced a similar artifact just before the Amelogenin marker. Figure 17. Sample dependent artifact (IDP_PET-1), obtained from 10 ng of pig bone marrow DNA, appearing at bp just before Amelogenin. The y-axis is scaled to 12,000 RFU and the artifact is listed with a peak height of 12,067 RFU. However, the artifact peak is so off-scale that the peak is splitting, resulting in an artificially low peak height. The peak appearing at bp in the NED dye channel is pullup from the off-scale artifact peak in PET. Conclusions Thermo Fisher Scientific s Human Identity STR kits are subjected to rigorous development specifications and criteria to prevent the presence of artifacts. However, the diversity of sample types, variation in storage conditions, and the use of analysis approaches that promote higher sensitivity for the forensic application can lead to the observation of artifacts not typically observed during development. This document will be periodically updated with additional reported and characterized artifacts identified post-product development to assist in the analysis and interpretation of Identifiler Plus STR results. 15

16 Comments *The observed percentages are provided for guidance only and are based on available data. These percentages have not been confirmed to the same extent as, for example, average marker stutter percentages. ** Refer to for information on BLAST statistics. BLAST statistics and matches observed can change over time as new sequences are added to the database. Match information and the associated statistics are provided for informational purposes only and do not suggest that a particular artifact was conclusively caused by the named organism. Revision History Revision Date Description A 3/23/2018 Initial publication. A.1 3/29/2018 Corrected Figure references for IDP_FAM-1, IDP_D8-1, IDP_vWA-1, IDP_vWA Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. 16