. 2021 Feb 9;10(1):53–74. doi: 10.3233/JHD-200433

Table 2.

Main characteristics of different methods for the preparation of libraries for MiSeq and PacBio for the sequencing of CAG repeats and the quantification of somatic mosaicism

Library preparation	Sequencing platform	Max CAG repeat size	Max modal CAG size^*	Max somatic expansion size	Estimated sequencing cost per sample^**	Quantity of genomic DNA required per sample	Indels	Relative accuracy of the quantification of somatic expansions	Relative accuracy of the quantification of somatic contractions
Bulk-PCR	Illumina MiSeq	∼115	∼90	∼120	∼$8^†	20 ng	No	+	not detected if
	PacBio RSII	≥550	∼250	≥550	∼$50^†	0.1 to 1.2 μg	Yes	+	not very large
	PacBio Sequel	≥550^SM	∼250^SM	≥550^SM	∼$30^†	20 to 240 ng^SM	Yes	+
	PacBio Sequel II	≥550^SM	∼250^SM	≥550^SM	∼$10^†	20 to 60 ng^SM	Yes	+
SP-PCR	Illumina MiSeq	∼115^SM	∼90^SM	∼120^SM	∼$8^‡	2 ng^SLM	No	++	+
	PacBio RSII	≥1500^L	≥1200^L	≥1500^L	∼$50^‡	10 to 120 ng^SLM	Yes	++	+
	PacBio Sequel	≥1500^LM	≥1200^LM	≥1500^LM	∼$30^‡	2 to 24 ng^SLM	Yes	++	+
	PacBio Sequel II	≥1500^LM	≥1200^LM	≥1500^LM	∼$10^‡	2 to 6 ng^SLM	Yes	++	+
Amplification-	Illumina MiSeq	∼115^S	∼90^S	∼120^S	∼$20^†	≥5μg^L	No	+++	+++
free	PacBio RSII	≥1500^L	≥150^L	≥1500^L	∼$1000^†	≥5μg^L	Yes	+++	+++
	PacBio Sequel	≥1500^LM	≥150^LM	≥1500^LM	∼$600^†	≥5μg^L	Yes	+++	+++
	PacBio Sequel II	≥1500^LM	≥150^LM	≥1500^LM	∼$100^†	≥5μg^L	Yes	+++	+++
Amplicon	Illumina MiSeq	∼115^S	∼90^S	∼120^S	∼$20^#	20 to 150 ng^L	No	+++	+++
sequencing	PacBio RSII	≥550^SM	∼250^SM	≥550^SM	∼$1000^#	2.5 to 30μg^SL	Yes	+++	+++
of barcoded	PacBio Sequel	≥550^SM	∼250^SM	≥550^SM	∼$600^#	0.4 to 4.8μg^SLM	Yes	+++	+++
single molecules	PacBio Sequel II	≥550^SM	∼250^SM	≥550^SM	∼$100^#	0.2 to 0.6μg^SLM	Yes	+++	+++

Observations described in this study are indicated in bold, the other information corresponds to expectations for approaches not used in this study based on observations described in this study (^S), on what has been described on other trinucleotide loci in the literature (^L) and/or on the manufacturer information available for each sequencing platform (^M). ^*: Max modal allele size for which the modal allele size can be estimated by sequencing and for which somatic mosaicism will reliably be quantified. ^**: based on [63]. †: assuming a minimum of ∼5,000 reads per sample and a maximum of 384 samples per sequencing run. ‡: assuming 20 SP-PCRs per sample and 250 reads per SP-PCRs. #: assuming ∼20 reads per single molecule and the genotyping of 5,000 single molecules.