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Forum: General 06-05-2019, 08:34 AM
Replies: 3
Views: 1,373
Posted By ATϟGC
I have tried the following protocol with insects...

I have tried the following protocol with insects and it works quite well in terms of purity and length:

Modified low‐salt CTAB extraction of high‐quality DNA from contaminant‐rich tissues
...
Forum: Sample Prep / Library Generation 05-24-2019, 08:32 AM
Replies: 6
Views: 947
Posted By ATϟGC
I tried to use limiting amount of SPRI beads...

I tried to use limiting amount of SPRI beads (Sera-Mag speedbeads and Commercial preparations diluted in their own buffer) for the normalization of amplicons prior to pooling without any success. I...
Forum: Illumina/Solexa 04-23-2019, 04:42 AM
Replies: 2
Views: 649
Posted By ATϟGC
A few questions that might help: What DNA...

A few questions that might help: What DNA polymerase are you currently using for PCR? Are your 16S primers tailed for high-throughput sequencing?

If inhibition is the problem, dilution will often...
Forum: Bioinformatics 04-02-2019, 05:07 AM
Replies: 7
Views: 667
Posted By ATϟGC
I will just echo cmbetts in saying that you...

I will just echo cmbetts in saying that you should always remove your oligo/primer sequences from Sanger and High-throughput sequencing data since they are artificial. You can sometimes find...
Forum: Illumina/Solexa 03-18-2019, 05:03 AM
Replies: 2
Views: 931
Posted By ATϟGC
Thanks Genomax. It is good to hear that it is...

Thanks Genomax. It is good to hear that it is comparable to other Illumina instruments and that dual indexes should mitigate the hopping.
Forum: Illumina/Solexa 03-13-2019, 06:24 AM
Replies: 2
Views: 931
Posted By ATϟGC
NovaSeq error rate?

I was wondering if anybody can quote the error rate for a NovaSeq? Even an idea of how it compares to MiSeq and HiSeq would be great as I can not seem to find any comparisons or hard numbers...
Forum: Sample Prep / Library Generation 11-22-2018, 10:11 AM
Replies: 4
Views: 975
Posted By ATϟGC
Since a lot of dsDNA fluorescent dyes have an...

Since a lot of dsDNA fluorescent dyes have an excitation maximum of about 490 nm (blue light) and and emission at about 520 nm (green light) You can make your own blue light epi-illuminator to excise...
Forum: Sample Prep / Library Generation 06-15-2018, 07:42 AM
Replies: 5
Views: 1,648
Posted By ATϟGC
If agarose gel electrophoresis is your only...

If agarose gel electrophoresis is your only option for fragment size distribution, GelStar is also very sensitive and might be more suitable than Sybr Gold for using in the gel itself.

Gelstar use...
Forum: Sample Prep / Library Generation 06-04-2018, 11:34 AM
Replies: 3
Views: 935
Posted By ATϟGC
Hi again Sean, Sorry for making that...

Hi again Sean,

Sorry for making that guess/assumption. I forgot that eukaryotes have 16S RNA as well.

So it does appear that you have very low base diversity, you have rapidly declining q...
Forum: Sample Prep / Library Generation 06-01-2018, 04:56 AM
Replies: 3
Views: 935
Posted By ATϟGC
Hi Sean, Aside from PhiX, are you taking any...

Hi Sean,

Aside from PhiX, are you taking any measures to increase diversity in your amplicon libraries such as staggering/offsetting and/or adding other loci?


At a glance, your cluster...
Forum: General 05-31-2018, 04:28 AM
Replies: 6
Views: 1,848
Posted By ATϟGC
You are very welcome. Sounds like over-drying is...

You are very welcome. Sounds like over-drying is likely your main problem. If you are worried about ethanol contamination and want to minimize drying time you can remove as much ethanol as you can...
Forum: General 05-30-2018, 05:40 AM
Replies: 6
Views: 1,848
Posted By ATϟGC
I find it can be hard to get more than 80-90%...

I find it can be hard to get more than 80-90% recovery and I sometimes get as low as 50%.

Here are a few suggestions you can try for improving recovery:

-Do not dry the beads too long. If you...
Forum: Illumina/Solexa 09-06-2017, 06:12 AM
Replies: 7
Views: 1,850
Posted By ATϟGC
Is that judged from the thumbnail images or the...

Is that judged from the thumbnail images or the densities as measure in the flow cell chart of SAV?
Forum: Illumina/Solexa 09-06-2017, 05:19 AM
Replies: 7
Views: 1,850
Posted By ATϟGC
You might want to compare the clustering at the...

You might want to compare the clustering at the top (where sample was loaded) versus the bottom.

With typical cluster generation there are more clusters at the top than the bottom of the lanes...
Forum: Sample Prep / Library Generation 07-04-2017, 11:52 AM
Replies: 9
Views: 2,005
Posted By ATϟGC
Hi SNPsaurus, I think that they are just...

Hi SNPsaurus,

I think that they are just referring to the typical single-digest RAD method where restriction is followed by sonication or another shearing method to generate fragment size...
Forum: Sample Prep / Library Generation 07-04-2017, 05:13 AM
Replies: 3
Views: 1,325
Posted By ATϟGC
I have always thought of the NaCl (or another...

I have always thought of the NaCl (or another source of Na+ like Sodium acetate) as a co-factor required for an ethanol, isopropanol or PEG-base nucleic acid precipitation. NaCl might precipitate...
Forum: Sample Prep / Library Generation 06-29-2017, 04:37 AM
Replies: 9
Views: 2,005
Posted By ATϟGC
Here is an article that may be of interest to you...

Here is an article that may be of interest to you if you are looking to decrease costs and improve sequence quality:
...
Forum: General 05-18-2017, 12:02 PM
Replies: 2
Views: 1,399
Posted By ATϟGC
As you have mentioned, plant DNA barcoding to the...

As you have mentioned, plant DNA barcoding to the species level is rather imperfect to put it mildly. Incomplete lineage sorting and hybridization have led to the sharing of chloroplast haplotypes in...
Forum: Sample Prep / Library Generation 03-29-2017, 08:28 AM
Replies: 4
Views: 3,530
Posted By ATϟGC
The best method might depend largely on the taxon...

The best method might depend largely on the taxon or taxa you are using if there are abundant co-precipitating contaminants. I have attached a method I developed/modified for HMW extraction of DNA...
Forum: Sample Prep / Library Generation 02-17-2017, 06:28 AM
Replies: 8
Views: 2,215
Posted By ATϟGC
If RNA contamination is your issue the following...

If RNA contamination is your issue the following likely won't help you, but it might if you problem is contaminants:

For recalcitrant DNA extractions I highly recommend trying an CTAB...
Forum: Sample Prep / Library Generation 02-08-2017, 10:37 AM
Replies: 8
Views: 1,801
Posted By ATϟGC
Hi again, I usually see poor nanodrop-Qubit...

Hi again,

I usually see poor nanodrop-Qubit correlations when the A260/230 get below ~1.7. Here is a link to another thread discussing common contaminants and their effects on the spectra of...
Forum: Sample Prep / Library Generation 02-08-2017, 04:49 AM
Replies: 8
Views: 1,801
Posted By ATϟGC
What type of samples are you extracting from? I...

What type of samples are you extracting from? I ask this because your poor correlations could also be due to co-precipitants like polysaccharides that give you poor A260/230 values. Ideally, A260/230...
Forum: MGISEQ (FKA Complete Genomics) 01-31-2017, 06:28 AM
Replies: 5
Views: 4,834
Posted By ATϟGC
I would expect that Sorbus species would have a...

I would expect that Sorbus species would have a fair amount of polysaccharides. I have had good success precipitating plant and animal DNA by CTAB dilution. Here are two articles with protocols:
...
Forum: Sample Prep / Library Generation 01-17-2017, 02:12 PM
Replies: 1
Views: 1,531
Posted By ATϟGC
Hi, The optimal loading concentration and...

Hi,

The optimal loading concentration and phiX % spike in will likely depend largely on the nucleotide sequence diversity at the start of your reads (on the HiSeq 2000 the first 12 bp is the most...
Forum: Sample Prep / Library Generation 08-19-2016, 08:19 AM
Replies: 14
Views: 8,943
Posted By ATϟGC
I'll echo ECO and melop in saying that I've tried...

I'll echo ECO and melop in saying that I've tried SPRI beads (Ampure and Seramag speedbeads) to clean up WGA products as well as genomic DNA from home-brew library prep and genomic DNA extraction...
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