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A technical comparison of current genotyping methods using next-generation sequencing of multiplex barcoded libraries. Adapted from Wang et al. (2012). Flavors of genotyping using next-generation sequencing of multiplex DNA-barcoded reduced-representation libraries.

Method Random shearing Size selection Fragment size Enzymes† Multiplexing level‡ Analysis tool(s) Reference
Multiplex shotgun genotyping No Yes Size selected MseI 96 (up to 384) Burrows-Wheeler alignment tool Andolfatto et al., 2011
Restriction association DNA sequencing (RAD-seq) Yes Yes Size selected SbfI 96 Custom Perl scripts Baird et al., 2008
Double digest RAD-seq No Yes Size selected EcoRI and MspI 48§ MUSCLE¶ Peterson et al., 2012
2b-restriction association DNA No No 33–36 bp BsaXI# NA†† Custom Perl scripts Wang et al., 2012
Genotyping-by-sequencing No No <350 bp ApeKI‡‡ 48 (up to 384) TASSEL§§ Elshire et al., 2011
Genotyping-by-sequencing – two enzyme No No <350 bp PstI and MspI 48 (up to 384) TASSEL Poland et al., 2012a
Sequence-based genotyping No Yes Size selected EcoRI and MseI 32 Burrows-Wheeler alignment tool and unified genotyper Truong et al., 2012
PstI and TaqI
Restriction enzyme sequence comparative analysis No Yes Size selected MseI NA¶¶ Burrows-Wheeler alignment tool and Samtools Monson-Miller et al., 2012
All of these approaches can use different enzymes. Shown are the enzyme(s) used in the initial study.
All of these methods have the possibility to increase the number of multiplexed samples using additional unique barcodes. The multiplex level as reported in the reference paper. Given in parenthesis are subsequent increases.
§Combinatorial barcoding is possible, placing a barcode on each end of the DNA fragment. Using a set of 48 adapter P1 barcodes and × 12 polymerase chain reaction (PCR) 2 indices it is possible to uniquely label 576 individuals (48 [adapter P1 barcodes] × 12 [PCR2 indices]). This method would require paired-end sequencing.
MUSCLE, multiple sequence comparison by log-expectation.
#Uses type IIB restriction endonucleases.
††NA, not applicable.
‡‡Has been successfully applied to using PstI and HindIII (E. Buckler and R. Elshire, personal communication, 2012).
§§TASSEL, trait analysis by association, evolution, and linkage.
¶¶96-plexing reported but unpublished.