Other acceptable sample type: saliva (please call 267-426-1447 for kits)
Specimen Preparation
This order is for the mother of a patient undergoing MitoGenome Seq + Del. Please provide detailed clinical history and features for both the proband and the mother (if submitting a sample).
Regarding Blood Specimens: Heparinized specimens, severely hemolyzed specimens, frozen, clotted or possibly commingled specimens, blood in non-sterile or leaky containers, mislabeled or inappropriately labeled specimens.
Do not heat, freeze or centrifuge blood before shipment. Refrigerate sample until shipment.
Please call 6-1447 with any questions regarding non-blood specimens.
Storage/Transport Temperature
For CHOP Phlebotomy: Samples can be collected throughout the week. Samples collected on weekends or holidays are held in Central Labs and sent to the Genomic Diagnostic Lab the following business day.
For External Clients: Refrigerate sample until shipment. Send the sample at room temperature with overnight delivery for receipt Monday through Friday, optimally within 24 hours of collection.
Please contact the lab (267-426-1447) with questions regarding non-blood specimens.
Volume Required
3 mL whole blood or 8µg DNA
Minimum Required
2 mL whole blood
Phlebotomy Draw
Yes
Ordering Recommendations
Order the Maternal MitoGenome Seq + Del to submit the proband's biological mother as a comparator. The order should be placed in the family member’s medical record, not the patient’s medical record. For more information contact the lab at 6-1447 or by sending an email to DGDGeneticCounselor@email.chop.edu.
Next generation sequencing: The mitochondrial genome is specifically enriched by amplifying the entire mtDNA as a single linear amplicon using a high-fidelity long-range PCR system (LR-PCR). LR-PCR products are purified using the AMPure XP beads and subjected to library preparation using SureSelect QXT followed by NGS on the Illumina NovaSeq 6000 system (150bp paired-end reads) with an average sequencing depth of 20,000X. Two different mtDNA LR-PCR products are sequenced for each proband to increase sensitivity and specificity. Only one amplicon is sequenced for an accompanying maternal relative specimen, if provided. Sequences are aligned to the revised Cambridge Reference Sequence (rCRS, NC_012920.1) [Andrews 1999, PMID: 10508508]. Variant calls, single nucleotide and small insertion/deletion heteroplasmy quantification and haplogroup determination are achieved using an in-house custom-built bioinformatics pipeline.
Deletion detection: An in-house screening algorithm based on read depth is used to call deletions in the mitochondrial genes. The heteroplasmy level of mtDNA deletions identified by NGS is assessed using droplet digital PCR (ddPCR). The ddPCR assay quantifies the fraction of non-deleted to full-length mtDNA molecules, which is used to infer the heteroplasmy level of mtDNA molecules carrying the deletion.
CPT Codes
N/A. Included as part of patient/proband test.
Collection
Collect
Collect whole blood in a purple top (EDTA) tube.
Other acceptable sample type: saliva (please call 267-426-1447 for kits)
Specimen Preparation
This order is for the mother of a patient undergoing MitoGenome Seq + Del. Please provide detailed clinical history and features for both the proband and the mother (if submitting a sample).
Regarding Blood Specimens: Heparinized specimens, severely hemolyzed specimens, frozen, clotted or possibly commingled specimens, blood in non-sterile or leaky containers, mislabeled or inappropriately labeled specimens.
Do not heat, freeze or centrifuge blood before shipment. Refrigerate sample until shipment.
Please call 6-1447 with any questions regarding non-blood specimens.
Storage/Transport Temperature
For CHOP Phlebotomy: Samples can be collected throughout the week. Samples collected on weekends or holidays are held in Central Labs and sent to the Genomic Diagnostic Lab the following business day.
For External Clients: Refrigerate sample until shipment. Send the sample at room temperature with overnight delivery for receipt Monday through Friday, optimally within 24 hours of collection.
Please contact the lab (267-426-1447) with questions regarding non-blood specimens.
Volume Required
3 mL whole blood or 8µg DNA
Minimum Required
2 mL whole blood
Phlebotomy Draw
Yes
Ordering
Ordering Recommendations
Order the Maternal MitoGenome Seq + Del to submit the proband's biological mother as a comparator. The order should be placed in the family member’s medical record, not the patient’s medical record. For more information contact the lab at 6-1447 or by sending an email to DGDGeneticCounselor@email.chop.edu.
Next generation sequencing: The mitochondrial genome is specifically enriched by amplifying the entire mtDNA as a single linear amplicon using a high-fidelity long-range PCR system (LR-PCR). LR-PCR products are purified using the AMPure XP beads and subjected to library preparation using SureSelect QXT followed by NGS on the Illumina NovaSeq 6000 system (150bp paired-end reads) with an average sequencing depth of 20,000X. Two different mtDNA LR-PCR products are sequenced for each proband to increase sensitivity and specificity. Only one amplicon is sequenced for an accompanying maternal relative specimen, if provided. Sequences are aligned to the revised Cambridge Reference Sequence (rCRS, NC_012920.1) [Andrews 1999, PMID: 10508508]. Variant calls, single nucleotide and small insertion/deletion heteroplasmy quantification and haplogroup determination are achieved using an in-house custom-built bioinformatics pipeline.
Deletion detection: An in-house screening algorithm based on read depth is used to call deletions in the mitochondrial genes. The heteroplasmy level of mtDNA deletions identified by NGS is assessed using droplet digital PCR (ddPCR). The ddPCR assay quantifies the fraction of non-deleted to full-length mtDNA molecules, which is used to infer the heteroplasmy level of mtDNA molecules carrying the deletion.
