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Search: Posts Made By: TonyBrooks
Forum: Vendor Forum 12-15-2015, 01:04 AM
Replies: 105
Views: 39,774
Posted By TonyBrooks
We've run exomes that clustered at 259k/mm2. The...

We've run exomes that clustered at 259k/mm2. The data still looked fine to us (92% >Q30, >90% alignment rates). The quality does begin to tail off when over-clustered though. 75bp are generally fine...
Forum: General 09-22-2015, 12:31 AM
Replies: 12
Views: 1,580
Posted By TonyBrooks
My guess would be 1. Especially if you had a 384...

My guess would be 1. Especially if you had a 384 well fluorescent plate reader. It works out at less than $0.50 per data point ($15 per sample).
Forum: Sample Prep / Library Generation 06-24-2015, 12:21 AM
Replies: 16
Views: 7,352
Posted By TonyBrooks
Below 100ng then HS, 100ng-1ug then BR. It's...

Below 100ng then HS, 100ng-1ug then BR.
It's also good practice to re-QC normalised samples too.
Forum: Epigenetics 05-11-2015, 06:42 AM
Replies: 3
Views: 1,184
Posted By TonyBrooks
FastQC...

FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) has a module that checks for adapter contamination. It looks for the universal adapter sequences (used in TruSeq RNA/DNA and...
Forum: Sample Prep / Library Generation 05-11-2015, 06:33 AM
Replies: 23
Views: 14,270
Posted By TonyBrooks
Here a a few tips that I've found through...

Here a a few tips that I've found through experience:
1) titrate your adapter to your sample input. We use 1µL of 15µL for every 500ng of DNA. For making homebrew adapters we follow the protocol in...
Forum: Vendor Forum 04-30-2015, 03:49 AM
Replies: 105
Views: 39,774
Posted By TonyBrooks
I'm pretty sure you can't do this as the software...

I'm pretty sure you can't do this as the software would surely check which kit version you are using and tell you it's incompatible.
It also looks like they changed the dyes, so the lasers would...
Forum: Vendor Forum 04-30-2015, 03:44 AM
Replies: 105
Views: 39,774
Posted By TonyBrooks
This upgrade really is a pain in the backside. I...

This upgrade really is a pain in the backside. I have no idea why Illumina couldn't make this back compatible and then use the RFID chips to determine whether to process the run as required.

I...
Forum: Vendor Forum 04-30-2015, 02:37 AM
Replies: 105
Views: 39,774
Posted By TonyBrooks
I asked the same question to Illumina Tech...

I asked the same question to Illumina Tech Support last week. Here's their response.

"Besides the changes in the software, there was a significant change in the v2 reagents. We changed the dyes of...
Forum: General 03-30-2015, 11:09 PM
Replies: 1
Views: 1,151
Posted By TonyBrooks
Trade in with Illumina for a MiSeq?

Trade in with Illumina for a MiSeq?
Forum: Vendor Forum 12-16-2014, 06:43 AM
Replies: 105
Views: 39,774
Posted By TonyBrooks
Data quality is definitely inferior to both the...

Data quality is definitely inferior to both the MiSeq and HiSeq.
It's quick though, and perhaps more suited for counting applications, such as RNA-Seq and ChIPSeq than variant calling.
The...
Forum: Illumina/Solexa 10-29-2014, 07:36 AM
Replies: 10
Views: 4,656
Posted By TonyBrooks
I'll summarise below The primers are as...

I'll summarise below

The primers are as follows. I won't put the P5/P7 sequences in here as the are (c) Illumina. You should be able to find them by Googling. Make sure you use the paired end P5 &...
Forum: Bioinformatics 09-23-2014, 03:01 AM
Replies: 4
Views: 2,839
Posted By TonyBrooks
I'm using a modified version of a script from...

I'm using a modified version of a script from Shingo Kikugawa
It's single thread, so takes a while. It's still quicker than manual zcatting.

I spoke to Illumina about this and a version of...
Forum: Illumina/Solexa 08-08-2014, 02:05 AM
Replies: 37
Views: 35,150
Posted By TonyBrooks
aatgatacggcgaccaccgagauctacac---------------------...

aatgatacggcgaccaccgagauctacac----------------------------tagagcatacggcagaagacgaac
aatgatacggcgaccaccgacaggttcagagttctacagtccgacgatc------tcacttcgtatgccgtcttctgcttg...
Forum: Illumina/Solexa 08-07-2014, 08:02 AM
Replies: 37
Views: 35,150
Posted By TonyBrooks
Your primers are not compatible with the MiSeq....

Your primers are not compatible with the MiSeq. On the HiSeq you can run single read or paired end flow-cells. There are subtle differences between the sequences on the grafted oligos in each flow...
Forum: Illumina/Solexa 07-16-2014, 01:24 AM
Replies: 43
Views: 22,151
Posted By TonyBrooks
Yes, although we needed to redesign the assay to...

Yes, although we needed to redesign the assay to use a different priming site.
Forum: Bioinformatics 06-25-2014, 12:52 AM
Replies: 4
Views: 2,839
Posted By TonyBrooks
I've spoken to Illumina tech support and they...

I've spoken to Illumina tech support and they have pointed out a possible bug in bcl2fastq v2.

"We identified a bug caused by thread handling that in certain cases caused errors in the order of...
Forum: Bioinformatics 06-19-2014, 07:11 AM
Replies: 4
Views: 2,839
Posted By TonyBrooks
Picard failure on NextSeq data

So, we've just begun to run some of our new NextSeq data through our standard pipelines and hit a snag. bcl2fastq still gives us fastq by index, read and lane, so the eight files for every sample are...
Forum: Illumina/Solexa 06-19-2014, 06:59 AM
Replies: 1
Views: 2,987
Posted By TonyBrooks
Yep. They've changed the way dual indexing is...

Yep. They've changed the way dual indexing is done on on the NextSeq. BP10 is the primer to read the i5 index and needs to be added to #18 for all dual index runs. Our FAS told us paired end...
Forum: Bioinformatics 06-04-2014, 06:56 AM
Replies: 8
Views: 2,141
Posted By TonyBrooks
See here for more info ...

See here for more info

http://seqanswers.com/forums/showthread.php?t=15356
Forum: Bioinformatics 06-04-2014, 06:24 AM
Replies: 8
Views: 2,141
Posted By TonyBrooks
I'm assuming these were sequenced on a HiSeq? The...

I'm assuming these were sequenced on a HiSeq? The spike at 4 cycles is most likely a phenomenon known as Bottom Middle Swath (or BMS in Illumispeak). The HiSeq attempts to find focus before scanning...
Forum: Sample Prep / Library Generation 06-02-2014, 02:37 AM
Replies: 3
Views: 1,336
Posted By TonyBrooks
I was thinking about trying a double...

I was thinking about trying a double normalisation too. First using the Bioanalyser and next by qPCR. Might try that for the next run.
We'll also work on automating qPCR set up to avoid any...
Forum: Sample Prep / Library Generation 05-30-2014, 07:55 AM
Replies: 3
Views: 1,336
Posted By TonyBrooks
Inconsitencies with qPCR results

I've begun quantifying all libraries with qPCR (Kapa kit) but I'm getting massive inconsistencies with the number of reads I get from samples in a multiplex pool.
I dilute each final library...
Forum: Sample Prep / Library Generation 05-30-2014, 02:50 AM
Replies: 6
Views: 4,488
Posted By TonyBrooks
On a related note, you can also mix and match...

On a related note, you can also mix and match reagents and chips. As long as the protocol you run on the machine matches the reagents you should be gold. You need to load the reagents according to...
Forum: Vendor Forum 05-22-2014, 04:26 AM
Replies: 105
Views: 39,774
Posted By TonyBrooks
I can confirm that the most you can get out of a...

I can confirm that the most you can get out of a 75 cycle kit is currently 92 cycles (76|8|8). This means you can't use the dark cycles for sequencing like you can on the MiSeq. You can register the...
Forum: Vendor Forum 05-16-2014, 11:08 AM
Replies: 105
Views: 39,774
Posted By TonyBrooks
Does this mean you can't do more than 75 cycles...

Does this mean you can't do more than 75 cycles in a 75 cycle kit. I confirmed with tech support that there are 25 additional cycles for dual indexing and we were told we could use those 100 cycles...
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