NEB NGS Library - Science topic

Regarding interpretation of sequence variants using ACMG rules.

Are ACMG rules PS3 and PP3 exclusionary?

In other words, if both PS3 and PP3 rules are fulfilled, can PP3 can be applied?

PS3 - Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

PP3 - Multiple lines of computational evidence support a deleterious effect on the gene or gene product -conservation, evolutionary, splicing impact, etc.

P.S - For me, they are not exclusionary.

Both myself and a colegue have recently tried this kit and failed to obtain any DNA. I have gone through tech support from NEB and taken on board their suggestions but still the results are completely negative. The same tissue sources produce good quality DNA with Qiagen genomic tips. I would not be persisting with this kit if it were not for the fact that we are trying to span very long repeat regions using the Oxford Nanopore set-up and this kit is the recommended extraction method for their ultra-long read library prep. Are there better alternative out there (I work with a range of tissues including insect, protozoan, annelid and mammalian sources - rarely do I get the luxuary of blood samples or pure tissue culture cells.

Many tahnks in advance for any and all help

Dear researchers

I am interested in applying some Bayesian inferential method to OMICS and NGS data in order to detect the causal relationship underlying a specific cellular system. Can you please suggest some sources or links that I can get this kind of data from?. Thanks for help in advance.

Dear all!

I do RIP experiment on Arabidopsis (antibody free, using himeric protein and coated magnetic beads). In my case I obtain as little as 1 ng of RNA from the complex.

For library preparation we have Truseq stranded total RNA (the amount should be >100 ng of total RNA). Or NebnextUltra II (which starts from 500 pg of gDNA)

We also have kit for pre-amplification of mRNA Mint.

How to be with this little amount and kits that I have?

I only see this resolution: to pre-amplificate my RNA using Mint(1-2 cycles) - fragmentate it - use Nebnext kit.

What do you think?

We are looking into liquid handling systems for pipetting qPCR plates (384 well) and for NGS library preps (amplicon seq). There are many machines on the market that are able to do that (Biomek FX/NX, Tecan Freedom Evo, Eppendorf Epmotion, Agilent Bravo, Felix CyBio, Perkin Elmer, Hamilton...). My question is which of the stated systems have the most simple, flexible and straight forward software to use for stated applications.

Thank you for sharing your experiences,

Filip

need a simple workflow for RNAseq data analysis (differential analysis) in galaxy and how to visualise the data.  Please help me to find a detailed user friendly protocol -in galaxy platform to analyse and visualize RNAseq data (differential analysis).

I am planning a potential RADSeq project (my first one). I've heard from some people that RADSeq requires quality DNA samples (very high molecular weight, very clean, high concentration)- specifically that a column-based kit like Quiagen (etc) must be used. Others say that RADSeq can be performed using DNA from 'homemade' extraction procedures like CTAB/ chloroform (no columns).  There are various suggestions on clean-up as well.

Have you had experience with RADSeq and if yes, what DNA extraction method did you use? How quality was your DNA (what size on a gel, and what wavelength spectrometer reading). I am specifically interested in plant material or other tough material that is either degraded or full of secondary compounds. Any advice you have on this topic is much appreciated!

I was preparing small RNA libraries for an NGS. For checking the results I did a cloning and test Sanger sequencing, where I ended up with the same sequence over and over again in two different samples (human and ctenophores). Can you think of any possibility this is reasonable besides that there is no sample but just contamination? A blast doesn´t reveal any annotations, but it might be a Kozak sequence (last part is always GACAGTT). Thanks in advance!!

Hello, 

I want to contact people interested in genomics of sea cucumbers. I am starting the process of digestion with restriction enzymes (ezRAD protocol) in order to obtain SNPs for population and interspecific analysis. I would like to know about your experiences in this, if somebody has recommendations, and if we could do collaborative work.

I would also like to know if someone is working on the assembly of the sea cucumber genome.

I am looking for a suitable NGS library prep method for RRS/GBS/RAD sequencing of degraded/low/template/ancient DNA. Do you have any good protocol available to genotype 5-10K SNPs in 100-200 inds reliably starting from already fragmented DNA? Otherwise a suitable SNP genotyping assay for such low qual DNA samples would be good. Thanks.

We are dealing with microbial diversity analysis from different hot springs of Gujarat, India. I am interested to participate workshop of NGS-ION TORRENT and NGS Data analysis by Bioinformatics tools in India.

If you have tried the New England Biolab kit for building Illumina NGS libraries, I would be very interested in your impressions.