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Search: Posts Made By: luc
Forum: RNA Sequencing Yesterday, 11:14 PM
Replies: 1
Views: 73
Posted By luc
How do you know? I would suggest to follow the...

How do you know? I would suggest to follow the library prep protocol all the way to the end and then assess the library or lack thereof.
Forum: Illumina/Solexa 02-07-2019, 06:16 PM
Replies: 1
Views: 171
Posted By luc
PCR-free Y-adapter libraries can migrate in funky...

PCR-free Y-adapter libraries can migrate in funky ways and can hide adapter dimers. In short they are difficult to QC.
I would suggest a diagnostic PCR with a small aliquot of the libraries:...
Forum: Introductions 01-24-2019, 06:19 PM
Replies: 2
Views: 628
Posted By luc
Hi msharik, A big hello to Indore: ...

Hi msharik,

A big hello to Indore: https://en.wikipedia.org/wiki/Indore
Forum: Sample Prep / Library Generation 01-21-2019, 09:14 AM
Replies: 0
Views: 373
Posted By luc
"random" enzymatic DNA fragmentation - how does it work?

Hi All,

almost all sequencing library preparation kit providers now offer kits promising "random" enzymatic DNA fragmentation that are not based on transposon tagmentation - adapters are ligated...
Forum: Core Facilities 01-19-2019, 06:26 PM
Replies: 3
Views: 528
Posted By luc
Bukowski is correct. RNA-seq library prep...

Bukowski is correct. RNA-seq library prep protocols (starting from RNA) are optimized for consistency to allow quantitative DGE analysis. Your cDNA prep protocol might not be.
Is your cDNA...
Forum: Sample Prep / Library Generation 01-14-2019, 08:51 AM
Replies: 15
Views: 13,435
Posted By luc
Yep, the enzymatic fragmentation products...

Yep, the enzymatic fragmentation products generated by kits from from several vendors do not require any end-repair.
Forum: 454 Pyrosequencing 01-14-2019, 08:46 AM
Replies: 5
Views: 4,825
Posted By luc
In the age of long read sequencing (PacBio,...

In the age of long read sequencing (PacBio, Nanopore) and linked reads (10X Genomics), "mate pair" sequencing is as obsolete as 454.
Forum: Sample Prep / Library Generation 01-11-2019, 03:46 PM
Replies: 3
Views: 481
Posted By luc
Nextflex adapters are available for DNA...

Nextflex adapters are available for DNA applications and for RNA-seq applications. The latter are merely dilutions of the former, but Bioo provides the concentration info somewhere.
If you have...
Forum: Sample Prep / Library Generation 01-10-2019, 09:59 PM
Replies: 3
Views: 481
Posted By luc
Check the concentration of the Nextflex RNA-seq...

Check the concentration of the Nextflex RNA-seq adapters on their web page; I have not memorized it but it will work.
Forum: Pacific Biosciences 01-07-2019, 06:03 PM
Replies: 5
Views: 737
Posted By luc
Nothing about the read metrics raises warning...

Nothing about the read metrics raises warning flags - they could certainly have generated longer library molecules though since they did not multiplex the library.

Is the genome size about what...
Forum: Illumina/Solexa 12-29-2018, 01:39 PM
Replies: 9
Views: 1,349
Posted By luc
I would suggest to check the index reads for this...

I would suggest to check the index reads for this particular library to see which base is affected.
Forum: General 12-24-2018, 12:44 PM
Replies: 3
Views: 683
Posted By luc
Hi Kujin, to my knowledge the Lohman...

Hi Kujin,

to my knowledge the Lohman protocol should work, as is, on the iSeq - as any other Truseq library design. We do not have an iSeq however. Do you want to optimize it for paired-end...
Forum: General 12-20-2018, 09:24 PM
Replies: 3
Views: 683
Posted By luc
Could you point us to your complete protocol? ...

Could you point us to your complete protocol? There are multiple Tag-seq protocols out there.
Likely your first amplification was already failing.
Your PCR cycles shows two consecutive stages at...
Forum: Sample Prep / Library Generation 12-17-2018, 11:31 PM
Replies: 4
Views: 543
Posted By luc
Randomprimer-dimers will be basically unavoidable...

Randomprimer-dimers will be basically unavoidable for your low, unmeasurable input.
If the oligos are a separate reagent, you could try to reduce their concentration.
Forum: Bioinformatics 12-17-2018, 11:25 PM
Replies: 3
Views: 465
Posted By luc
bcl2fastq will only transfer the UMI information...

bcl2fastq will only transfer the UMI information into the read header and trim them out of the reads as an option - nothing else.
UMI-tools are more sophisticated and have many additional functions.
Forum: Illumina/Solexa 12-12-2018, 06:11 PM
Replies: 1
Views: 462
Posted By luc
Did you use custom sequencing primers for any of...

Did you use custom sequencing primers for any of the reads?
Forum: Sample Prep / Library Generation 11-06-2018, 11:36 PM
Replies: 2
Views: 807
Posted By luc
Both column kits and SPRI beads will work just...

Both column kits and SPRI beads will work just fine. Beads make it easier to work with multiple samples in parallel. Almost "All" manufacturers are now selling SPRI beads. I would bet they all work...
Forum: Illumina/Solexa 10-13-2018, 11:05 AM
Replies: 2
Views: 730
Posted By luc
As long as the short library molecules are a...

As long as the short library molecules are a smaller fraction of the loaded libraries (<20%?) there will be no problem.
Forum: Illumina/Solexa 10-10-2018, 10:30 PM
Replies: 1
Views: 751
Posted By luc
You are correct about the truseq style adapters....

You are correct about the truseq style adapters. Sorry I do not know about additional documentation.
Forum: Illumina/Solexa 10-08-2018, 05:50 PM
Replies: 1
Views: 610
Posted By luc
To be able to add single-T-tailed adapters like...

To be able to add single-T-tailed adapters like the Illumina Truseq adapters via ligation. This is a way to prevent (or strongly reduce) insert-to-insert ligation and adapter-to-adapter ligation.
Forum: General 10-03-2018, 11:30 AM
Replies: 13
Views: 1,895
Posted By luc
Thanks for the links!

Thanks for the links!
Forum: General 09-30-2018, 10:07 PM
Replies: 13
Views: 1,895
Posted By luc
I am not working in this field, but to me these...

I am not working in this field, but to me these ideas have some charm. I am curious about any supporting data.
Forum: Illumina/Solexa 08-05-2018, 04:46 PM
Replies: 2
Views: 978
Posted By luc
The 5' bias is mostly due to the A-tailing...

The 5' bias is mostly due to the A-tailing reaction.
Forum: Metagenomics 08-02-2018, 05:30 PM
Replies: 2
Views: 2,469
Posted By luc
What type of results do you have at the moment? ...

What type of results do you have at the moment?

I am sure there will be a way to generate heatmaps in Excel.

It will certainly better in the long run to become familiar with R/RStudio and the...
Forum: Illumina/Solexa 08-01-2018, 12:55 PM
Replies: 11
Views: 1,534
Posted By luc
I would certainly filter the reads based on...

I would certainly filter the reads based on average quality scores (not losing more than
15 % of the reads) and do some very gentle quality trimming from the 3' end.
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