Recovery of Whole Mitochondrial Genome From Compromised Samples Via Multiplex PCR and Massively Parallel Sequencing

Figures & data

Table 1.  Summary of relevant information and data collected for each sample.

Sample ID	Additional source information	mtGenome copy input	Average coverage	Percentage genome ≥100× coverage	Mitochondrial haplogroup
Challenging samples
Hair from dust bunny
DBH-1	Living room (2015)	11,351	6177	100	H7c4
DBH-2	Bedroom (2008)	8592	8251	100	H11a1
DBH-3	Bathroom (2008)	2172	5396	100	C1b2^†
DBH-4	Living room (2008)	4083	10,803	87	H7c4^†
DBH-5	Bedroom (2014)	5362	10,512	100	H1b1(T16362C)
Bone
FEM	Mid-shaft femur	3512	3251	96	U2b2^†
Cremated remains
R-1.1	Bone fragment	294,298	5092	98.6	U5b1c2^†
R-1.2	Bone fragment	197	16,887	97.9	U5b1c2^†
R-1.A	Ashes	1.54E6	14,721	99.1	U5b1c2^†
Maggot
MAG	FOREst donor	771,500	2691	58.4	H2a2a2^†
Single cell
FC-1	Buccal – female	N/A	1481	84.2	J1c4^†
MC-1	Buccal – male	N/A	455	60.4	L2a1a2^†
Reference samples
Hair
DH-1.1	Donor 1 (root)	12,911	20,676	100	J1c3e2
DH-1.2	Donor 1 (mid-shaft)	3179	13,603	100	J1c3e2
DH-2.1	Donor 2 (root)	6087	10,488	97.7	J1b1a1a
DH-2.2	Donor 2 (mid-shaft)	2205	1293	96.3	J1b1a1a
Buccal swab
DBu-1	Donor 1 (swab)	4.07E7	5630	100	J1c3e2
DBu-2	Donor 2 (swab)	5.06E6	5063	97.6	J1b1a1a

Source information includes naming conventions, date, location and method of collection where applicable. mtGenome copy input indicates number of mtGenome equivalents input into each multiplex PCR. MPS metrics include average coverage across the mtGenome and percentage of the mtGenome covered by at least 100 reads. Mitochondrial haplogroup is the haplogroup assigned by the program mthap.

† Indicates imperfect match from assigned haplogroup.

DBH: Hair shaft collected from dust bunny; DBu: Reference donor buccal swab; DH: Reference donor hair shaft; FC: Single female buccal cell; FEM: Femor; FOREst: Forensic Osteology Research Station; MAG: Maggot; MC: Single male buccal cell; MPS: Massively parallel sequencing; mtGenome: Mitochondrial genome; R: Cremated remains.

Figure 1.  Orientation of 46 primers around the mitochondrial genome.

Primers were multiplexed into three reactions (I, II, III). 43 primers were modified from Applied Biosystems MitoSEQr Kit (Thermo Fischer, MA, USA); starred (*) primers were separately redesigned.