Procedure for generating Illumina-compatible DNA libraries
Samples consisting of longer DNA fragments are first sheared into a random library of fragments that are a median fragment size of 300 bp (when using the Illumina MiSeq instrument, version 1), or 500 bp (when using the Illumina MiSeq instrument, version 2), in length. Following fragmentation, the ends of the DNA fragments are repaired and an A-overhang is added at the 3'-end of each strand. Afterwards, adaptors, which are necessary for amplification and sequencing, are ligated to both ends of the DNA fragments. Barcode adapters, which contain a unique identifying sequence, are also available with the GeneRead Library Prep (I) Kit and enable multiplex sequencing reactions to be performed. Fragments are then size selected and purified. Cleanup and size selection can be done using the GeneRead Size Selection Kit (cat. no. 180514), which allows precise, column-based size selection of DNA (see figure
Precise size selection). To ensure maximum yields from minimum amounts of starting material, an optional high-fidelity amplification step can also be performed.
Procedure for generating Ion Torrent-compatible DNA libraries
Samples consisting of longer DNA fragments are first sheared into a random library of fragments that are a median fragment size of 400 bp (when using the Ion Torrent PGM instrument), or 200 bp (when using the Ion Proton instrument), in length. Following fragmentation, the ends of the DNA fragments are repaired. Afterwards, adaptors, which are necessary for amplification and sequencing, are ligated to both ends of the DNA fragments. Barcode adapters, which contain a unique identifying sequence, are also available with the GeneRead Library Prep (L) Kit and enable multiplex sequencing reactions to be performed. Fragments are then size selected and purified. To ensure maximum yields from minimum amounts of starting material, an optional high-fidelity amplification step can be performed.
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