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Forum: Illumina/Solexa 04-16-2019, 12:09 AM
Replies: 18
Views: 6,276
Posted By JakobHedegaard
I am working in a hospital lab and am the happy...

I am working in a hospital lab and am the happy user of two Sciclone NGS, a NGS Express and a Zephyr NGS. We use the robots for DNA cleanup, NGS lib prep, purification and QC.
There are a number of...
Forum: Illumina/Solexa 03-29-2019, 02:01 AM
Replies: 2
Views: 437
Posted By JakobHedegaard
I have received the following clarifying reply...

I have received the following clarifying reply from Illumina Techsupport:
"The run number assignment is sequential to which side actually starts first after the pre-run checks. Usually FCA will have...
Forum: Illumina/Solexa 03-25-2019, 02:05 AM
Replies: 2
Views: 437
Posted By JakobHedegaard
Numbering of simultaneously started NovaSeq runs?

How are simultaneously started NovaSeq runs actually numbered?

First time we tried, the A-run was assigned with the lowest number. This would somehow be the logical way of numbering....
Forum: Sample Prep / Library Generation 03-11-2019, 03:59 AM
Replies: 2
Views: 806
Posted By JakobHedegaard
Hi Arctan, We have also done some bacterial...

Hi Arctan,
We have also done some bacterial RNA-Seq using Ribo-Zero (and ScriptSeq) and have observed the same peak. I have "only" generated the data, but have not received any feedback from the...
Forum: Sample Prep / Library Generation 01-30-2019, 06:01 AM
Replies: 17
Views: 6,310
Posted By JakobHedegaard
"To check if the large fragments are actually...

"To check if the large fragments are actually overamplification?"
Two tests to do:
1) denature a small aliquote of the library and run it on e.g. a Bioanalyzer. If the large peak is due to...
Forum: Illumina/Solexa 01-18-2019, 01:18 AM
Replies: 9
Views: 2,092
Posted By JakobHedegaard
In our hands, we do not find any significant...

In our hands, we do not find any significant differences in the quality between insert data with perfect index reads and insert data with 1-mismatch index reads.
So despite a much larger fraction of...
Forum: Illumina/Solexa 01-15-2019, 04:12 AM
Replies: 9
Views: 2,092
Posted By JakobHedegaard
And in a recent S2 (2x150) NovaSeq run of eight...

And in a recent S2 (2x150) NovaSeq run of eight Nextera DNA Flex libraries prepared from human gDNA using indexes from the CD Indexes (96 Indexes, 96 Samples, cat# 20018708) we observed a single...
Forum: Illumina/Solexa 01-15-2019, 04:03 AM
Replies: 9
Views: 2,092
Posted By JakobHedegaard
I have been told that two TruSeq UD indexes have...

I have been told that two TruSeq UD indexes have shown lower than expected perfect index reads: UDI0025 (ACTAAGAT, AACCGCGG) and UDI0055 (AGGCAGAG, AGAATGCC).
Anyone else seen this?
Forum: Sample Prep / Library Generation 01-10-2019, 01:01 AM
Replies: 2
Views: 1,257
Posted By JakobHedegaard
Take a look at...

Take a look at https://ls.beckmancoulter.co.jp/files/appli_note/Gel_Free_Using_SPRIselect.pdf
But I agree with nucacidhunter that 50 bp is a too narrow window for a double-SPRI
Forum: Illumina/Solexa 12-20-2018, 12:24 AM
Replies: 9
Views: 2,092
Posted By JakobHedegaard
Come on - I have heard several rumors about...

Come on - I have heard several rumors about indexes behaving non-optimal on the NovaSeq. Please share your experiences :-)
Forum: Illumina/Solexa 12-19-2018, 01:57 AM
Replies: 9
Views: 2,092
Posted By JakobHedegaard
NovaSeq index issue (high fraction of non-perfect indexes)

Hi,
We have conducted a NovaSeq S1 run of different library types and noticed a lower %perfect reads from a single sample as compared to a MiSeq QC run of the same libraries.
The libraries were a...
Forum: Sample Prep / Library Generation 08-27-2018, 01:08 AM
Replies: 4
Views: 1,498
Posted By JakobHedegaard
Hi Whe, Have you considered a Sciclone NGS,...

Hi Whe,
Have you considered a Sciclone NGS, Zephyr NGS og Janus NGS Express from Perkin Elmer? See...
Forum: Illumina/Solexa 07-24-2018, 11:21 PM
Replies: 6
Views: 1,596
Posted By JakobHedegaard
We have been using Ribo-Zero + ScriptSeq for...

We have been using Ribo-Zero + ScriptSeq for years with success. Primarily for samples of human RNA (using Ribo-Zero GOLD or Globin-GOLD) but also a few samples of bacterial RNA (of course using the...
Forum: Sample Prep / Library Generation 05-18-2018, 02:03 AM
Replies: 0
Views: 871
Posted By JakobHedegaard
gDNA library prep kit for NovaSeq 6000?

Going to have a NovaSeq installed soon and wonder which library prep kits are most optimal for human gDNA? We are going to use the libraries for both WGS and for capture (exome/panels). Index hoping...
Forum: Illumina/Solexa 11-22-2017, 10:08 PM
Replies: 12
Views: 2,111
Posted By JakobHedegaard
A update on the chemistry-only recipe.... We...

A update on the chemistry-only recipe....
We got a custom recipe from Illumina (without any guarantee) but it didn't work. It was supposed to do chemistry only in cycle 13-17 - but it didn’t.
/Jakob
Forum: Illumina/Solexa 10-26-2017, 01:14 AM
Replies: 13
Views: 1,767
Posted By JakobHedegaard
See the file /Reports/html/index.html in the run...

See the file /Reports/html/index.html in the run folder. It includes a list of top10 unknown barcodes (and the known ones). Click on "show barcodes" in the top-right corner when you have opened the...
Forum: Sample Prep / Library Generation 10-23-2017, 05:42 AM
Replies: 1
Views: 977
Posted By JakobHedegaard
Hi, If we happen to have libraries of low...

Hi,
If we happen to have libraries of low concentration, we denature with 0.2 M NaOH, add an equal volume of 200 mM Tris-HCl, pH 7, and adds HT1 to reach 20 pM.
An example: a library is 230 pM....
Forum: Illumina/Solexa 10-19-2017, 11:34 PM
Replies: 2
Views: 921
Posted By JakobHedegaard
Hi kerryp, No problem. Have done it...

Hi kerryp,
No problem. Have done it successfully for several years doing Nextera XT libraries of bacteria and sequencing on NextSeq medium in batches of 50-70 bacteria.
I havent had concerns about...
Forum: Illumina/Solexa 10-18-2017, 11:57 PM
Replies: 12
Views: 2,111
Posted By JakobHedegaard
Hi Provirus, Please send me a privat message. ...

Hi Provirus,
Please send me a privat message.
/Jakob
Forum: Illumina/Solexa 10-18-2017, 10:14 PM
Replies: 12
Views: 2,111
Posted By JakobHedegaard
Hi Provirus and Austinso, What a coincidence....

Hi Provirus and Austinso,
What a coincidence. We have just been in contact with Illuminas Techsupport to have a custom program made for the NextSeq 500 to do a PE run with 5 dark cycles after the...
Forum: Illumina/Solexa 08-22-2016, 10:45 PM
Replies: 2
Views: 2,322
Posted By JakobHedegaard
Hi, thank you very much for sharing this. I...

Hi, thank you very much for sharing this.
I have made a simple and a bit rough R-script (attached) to compare two list of indexes and return a similarity matrix, which can be used to select index...
Forum: Sample Prep / Library Generation 07-27-2016, 01:36 AM
Replies: 0
Views: 787
Posted By JakobHedegaard
LabChip GX - daisy chains?

Hi,
We are happy using a LabChip GX for our NGS work - both QC of gDNA and of libraries.
But we have an issue with daisy-chains in NGS libraries which seem to be related to the GX and not the...
Forum: Sample Prep / Library Generation 02-19-2016, 02:29 AM
Replies: 1
Views: 827
Posted By JakobHedegaard
Degraded RNA from bacteria biofilms following Ribo-Zero

Hi,
We have applied Ribo-Zero to two sets of RNA isolated from bacteria. Set A was isolated from bacteria grown as biofilm and set B was from bacteria in culture. The total RNA is fine from both...
Forum: Sample Prep / Library Generation 01-27-2016, 05:29 AM
Replies: 1
Views: 878
Posted By JakobHedegaard
Hi again, I have made a small R script...

Hi again,
I have made a small R script (attached, "compare_index_Rscript.txt") to compare two lists of indexes (equal index length) and return the number of different positions per combination -...
Forum: Sample Prep / Library Generation 01-27-2016, 02:15 AM
Replies: 1
Views: 878
Posted By JakobHedegaard
Selection of indexes

Hi,
How to select an optimal set of indexes from different library prep kits to design a sequence run?
An example: I am about to prepare libraries and sequence 40 samples prepared with XT dual...
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