Table 1.

Principles, outcomes, and challenges with techniques used to study immune repertoires in multiple sclerosis

Technology	Principle	Outcome	Challenges
Southern blot	DNA is digested by restriction enzymes, separated on agarose gels, and blotted onto nitrocellulose membranes. DNA fragments are hybridized with gene probes.	Clonal composition and diversity	Requires relatively large amounts of DNA Does not give the nucleic acid sequence Low throughput
CDR3 spectratyping	T‐cell receptor cDNA is amplified by PCR across the CDR3 region using primers mapping to different families of variable genes. Since T‐cell clones differ in CDR3 length, the distribution of lengths of the resultant PCR products reflects the overall diversity. Deviations from a bell‐shaped distribution indicate clonal expansions.	Overview of the clonal composition and diversity	Limited resolution Does not give the nucleic acid sequence
Flow cytometry based T‐cell receptor (TCR) Vβ repertoire analysis	A cell sample is stained with antibodies against different TCR‐Vβ‐families and analyzed on a flow cytometer.	Overview of the clonal composition	Limited resolution Only available for T cells and only for β‐chain families Does not give the nucleic acid sequence
Sanger sequencing	The sequencing technique is based on selective incorporation of chain‐terminating dideoxynucleotides. The resulting DNA fragments are separated by electrophoresis.	Nucleic acid sequence, up to 700 bp	Low‐throughput limits the capacity for assessing repertoire diversity
High‐throughput sequencing technologies
Roche 454	“Sequencing by synthesis,” based on the release of pyrophosphate on nucleotide incorporation.	Nucleic acid sequence, 400 bp (recently upgraded to 1000 bp)	Prone to insertions and deletions (“indels”), which cannot be distinguished from true insertions and deletions within CDR3
Illumina (HiSeq and MiSeq)	“Sequencing by synthesis,” based on cyclic reversible termination, which is an adaption of Sanger sequencing. “Paired end” sequencing makes it possible to sequence both ends of a fragment, and subsequently align the reads to cover longer sequences.	Nucleic acid sequence, 2 × 150 bp for HiSeq and 2 × 300 bp for MiSeq	Due to shorter read length, HiSeq does not cover the entire immunoglobulin heavy‐chain variable and therefore not all somatic mutations