Depletion of repetitive sequences – WGS libraries

As you know, the sunflower genome contains a large amount of repetitive sequences, that is why it is so big and so annoying to sequence. I have been working for a while on optimizing a depletion protocol, to try to get rid of some repetitive sequences in NGS libraries (transposons, chloroplast DNA…). Continue reading

Making Illumina Whole Genome Shotgun Sequencing Libraries – (Dan E.)

I’ve been making whole genome shotgun sequencing libraries (for the purposes of this post: WGSS libraries) to sequence sunflower genomes on the Biodiversity Centre’s Illumina HiSeq. I haven’t been doing it for very long and its likely that my approach will change in the future as costs and products change but, as of early 2012, I’ve landed on a hybrid protocol based on kits from an outfit called Bioo Scientific. I use the Bioo Sci. adapter kit and their library prep kit up to the final PCR step at which point I switch to a PCR kit from another outfit called KAPA. I also use a KAPA kit to quant libraries with qPCR. In this post I give a little context then describe what I do to make WGSS libraries . . . Continue reading

Illumina Sequencing Adapters and Barcodes (Dan E.)

As of March 2012 we are using the Bioo Scientific NEXTflex barcoded adapters for WGS sequencing libraries made by ourselves, (well me so far). The set we are currently using comprises 48 barcodes, so we can multiplex up to a 48-plex in one lane on the Illumina HiSeq sequencer.

Bioo Sci. 48 barcoded adapters

Below are the sequences of the Illumina adapters and the 48 barcodes we are currently using. Continue reading