How to do effective SPRI beads cleaning

SPRI beads cleaning is one of the most repetitively used step during library preparations and probably the step where most of us lose a lot of precious DNAs.

Losing DNA scared me so much (because it can be observable) that I hesitated a lot before trying to use beads to concentrate genomic DNA, because usual rate of recovery are ~50%.

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Hopefully with some practice and a lot of patience, it is possible to reach 90% recovery. How to lose as little DNA as possible? Here are some guidelines: Continue reading

Home-brew WGS library multiplexing

There are two main ways to barcode WGS libraries so that they can be run together on a same lane:

– In-line barcodes: unique sequences are located at the very end of one or both adapters. This sequence will be at the very beginning of each read from a given library. This is the barcode system that is normally used fro GBS libraries as well.

– Indices: barcodes are in the middle of one or both adapters. These barcodes are read through an independent round of sequencing. For a paired-end library you would have therefore two rounds of sequencing of your fragment and a third round of sequencing for the index (and I guess a fourth one as well, if you have double indices). This is the system used in most commercial kits.

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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

(Probably the closest you can get to) Home-brew Illumina WGS libraries

As some of you might know, I have been working for the last few months on optimizing a protocol for Illumina WGS libraries that will reduce our dependency on expensive kits without sacrificing quality. The ultimate goal would be to be able to use WGS libraries as a more expensive but hopefully more informative alternative genotyping tool to GBS. Getting to that point ideally requires to develop:

1) A cheaper alternative for library preparation (this post)

2) A reliable multiplexing system (this other post)

3) A way to shrink the sunflower genome before sequencing it (because, as you know, it’s rather huge) (yet another post)

The following protocol is for non-multiplexed libraries. The protocol for multiplexed ones is actually identical, you just need to change adapters and PCR primers – more about that in the multiplexing post.

If you are planning to pool libraries and deplete them of repetitive elements, read carefully all three posts before starting your libraries (mostly because you might need to use different adapters and PCR primers)

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NEB HF enzymes – regarding library preps

Hi guys,

Just a note about PstI-HF:

The PstI-HF sold from NEB is less processive than the old NEB PstI/red stripe version (go here to see it: https://www.neb.com/products/r0140-psti) .  When digesting ultrapure, high concentration homogeneous DNA (plasmids) digestions fail to go to completion even when left overnight (> 16 hours).  What does this mean for you?  It means that your genomes which are more difficult substrates to deal with will not digest fully unless left to go overnight.  If you want to ensure a good digest use a positive control and switch to the linked product.

 

 

 

 

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

Shearing DNA for WGS Sequencing- Bioruptor (Dan E.)

This post is about fragmenting, or shearing, genomic DNA to a particular size range using the Rieseberg lab’s Bioruptor sonicator.

Most of the current whole genome shotgun (WGS) library preparation protocols for NGS applications start with fragmented DNA. Generally speaking, this starting DNA should be a certain size and, for multiple samples, consistently that size. This objective turns out to be quite a tricky thing to accomplish with the Bioruptor. Given that WGS sequencing will probably continue to be popular in the lab, I am posting here what I have learned so far about taking whole genomic sunflower DNA and smashing it to the size range that I want using the Bioruptor. If I discover anything else in future library preps I’ll add it below. If anybody else has useful tips please comment.

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