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Forum: Illumina/Solexa 08-22-2016, 12:10 PM
Replies: 1
Views: 1,160
Posted By MLog
script for checking barcode compatibility

Hi everyone,

a colleague of mine wrote a script that checks the compatibility of barcodes used in different sample prep kits for Illumina (Truseq LT/HT, Nextera, Nugen) - for the case when we...
Forum: Sample Prep / Library Generation 04-30-2015, 02:17 PM
Replies: 7
Views: 4,838
Posted By MLog
Hi Simone78, do you have any examples of...

Hi Simone78,

do you have any examples of Bioanalyzer traces of RNA samples after SD-RNA depletion? I tried this protocol (as implemented in NEBNext rRNA removal kit) and got some strange results -...
Forum: Sample Prep / Library Generation 06-11-2014, 02:37 PM
Replies: 5
Views: 4,359
Posted By MLog
I usually take 1-3 micrograms of total RNA. With...

I usually take 1-3 micrograms of total RNA. With human and bacterial RNA it worked well - less than 1% of reads mapped to rRNA. Probably for Drosophila it's worth trying to take less.
Forum: Sample Prep / Library Generation 06-10-2014, 10:04 PM
Replies: 5
Views: 4,359
Posted By MLog
Hi Wei, not as good as I wish, actually - about...

Hi Wei,
not as good as I wish, actually - about 30-40%. Next time I did double depletion and it worked much better, with ~ 7% mapped ro rRNA.
Forum: Sample Prep / Library Generation 03-07-2014, 02:05 PM
Replies: 4
Views: 1,666
Posted By MLog
"material is coming from M.lignano" - Do you mean...

"material is coming from M.lignano" - Do you mean Macrostomum lignano, a flatworm? I recently extracted RNA from another flatworm, Opistorchis, and it looks exactly the same.
THis is actually not...
Forum: Sample Prep / Library Generation 03-07-2014, 01:47 PM
Replies: 5
Views: 4,359
Posted By MLog
ribosomal RNA depletion

Hello,
I'm using Epicentre Ribo-Zero kit for removal of rRNA from Drosophila. I checked RNA on Agilent, before and after depletion, and I observed a picture that seems strange for me (attached). A...
Forum: Illumina/Solexa 12-08-2013, 02:32 PM
Replies: 4
Views: 3,105
Posted By MLog
No, this is not a contamination between runs, if...

No, this is not a contamination between runs, if you mean this.
Forum: Illumina/Solexa 12-08-2013, 01:42 PM
Replies: 4
Views: 3,105
Posted By MLog
Question Contamination between samples run on the same lane

Hi colleagues,
I found a strange effect when sequencing several samples on Miseq. The reads from each sample contain about ~0.1% of reads from other samples run on the same flowcell. This does not...
Forum: Illumina/Solexa 11-25-2013, 02:42 AM
Replies: 15
Views: 15,166
Posted By MLog
If the flowcells are the same for v. 3 and v. 2,...

If the flowcells are the same for v. 3 and v. 2, this should mean that it is possible to get 25M reads using v.2 kit, isn't it? But this doesn't seem to be the case, unfortunately.
Forum: Illumina/Solexa 10-09-2013, 06:31 AM
Replies: 26
Views: 14,339
Posted By MLog
sorry for such strange question, but what is the...

sorry for such strange question, but what is the colour of incorporation mix in Miseq kits v.3?
I got our first v.3 kit today and I'm very much surprised to see that incorporation mix is dark blue,...
Forum: Bioinformatics 06-18-2013, 11:20 PM
Replies: 3
Views: 844
Posted By MLog
Actually, we first sequenced on Hiseq several...

Actually, we first sequenced on Hiseq several samples of different individuals (treated and non-treated, 8 in total, ~ 25 millions of reads per sample) and tried to assemble then all together. After...
Forum: Illumina/Solexa 06-13-2013, 10:33 AM
Replies: 24
Views: 6,908
Posted By MLog
I checked the flowcell, it looked perfectly OK...

I checked the flowcell, it looked perfectly OK and was delivering proper volumes of water during washes. So we retried with another kit and it worked without any problems. Looks like this no clusters...
Forum: Bioinformatics 06-13-2013, 10:25 AM
Replies: 3
Views: 844
Posted By MLog
Expression analysis using non-assembled reads?

Hi,

does everyone heard about assembly-free/reference-free expression analysis tools?

We have a species with no reference available and the idea is to study gene expression changes after some...
Forum: Illumina/Solexa 06-09-2013, 11:53 PM
Replies: 24
Views: 6,908
Posted By MLog
Hi memento, have you found what was the...

Hi memento,

have you found what was the reason of failed run? Was this an instrument issue or rather reagents/library?
I'm asking because I got the same error from our Miseq last week. If this is...
Forum: Sample Prep / Library Generation 06-04-2013, 09:13 PM
Replies: 35
Views: 18,519
Posted By MLog
I used the same program as for standard TruSeq...

I used the same program as for standard TruSeq RNA kit (50 minutes at 42C). I will sequence these libraries soon and will write here about results.
Forum: Sample Prep / Library Generation 05-30-2013, 12:06 AM
Replies: 35
Views: 18,519
Posted By MLog
Hi jazz, indeed, the enzyme used in this kit...

Hi jazz,
indeed, the enzyme used in this kit is user-supplied SuperScript II, as kcchan already mentioned. So 15 minutes looks like a typo.
As for the method, it seems that Illumina uses the...
Forum: Sample Prep / Library Generation 05-29-2013, 03:13 AM
Replies: 35
Views: 18,519
Posted By MLog
Question Hi all, I started to use Illumina TruSeq...

Hi all,
I started to use Illumina TruSeq Stranded mRNA kit: http://support.illumina.com/sequencing/sequencing_kits/truseq_stranded_mrna_lt_sample_prep_kit.ilmn
and I noticed a couple of thing that...
Forum: De novo discovery 05-12-2013, 03:28 PM
Replies: 4
Views: 1,847
Posted By MLog
Hi benR, just in case if you are still...

Hi benR,

just in case if you are still following this thread...
I don't know the reason, but I observe similar bias in most (if not all!) our DNA libraries (example attached). We are using Truseq...
Forum: Sample Prep / Library Generation 05-10-2013, 05:00 AM
Replies: 10
Views: 3,812
Posted By MLog
Hi ychang (and everyone who has experience with...

Hi ychang (and everyone who has experience with Nextera MP, too),
did you already sequenced and mapped these libraries? I wonder how many reads mapped with expected insert size?
I tried Nextera MP...
Forum: Illumina/Solexa 05-01-2013, 11:32 AM
Replies: 8
Views: 4,637
Posted By MLog
When they say 374 million/lane, they mean 374...

When they say 374 million/lane, they mean 374 millions of 100bp reads, or 187 millions of 100+100 reads. This gives ~ 37 Gb. In our hands, we usually get 30-40 Gb, with Q30 ~90%.
It is possible to...
Forum: Illumina/Solexa 04-28-2013, 05:54 AM
Replies: 4
Views: 1,579
Posted By MLog
Certainly yes - agilent or agarose...

Certainly yes - agilent or agarose electrophoresis. (Though actually Agilent sometimes generates weird results). I think this bias could a be result of short inserts, those that are shorter than the...
Forum: Illumina/Solexa 04-27-2013, 02:33 PM
Replies: 4
Views: 1,579
Posted By MLog
Hi induivain, I observe exactly the same...

Hi induivain,

I observe exactly the same effect (even stronger, example attached) on my 250-bp runs. My example is from a recent run of an exome sample, but it seems that this bias is not related...
Forum: Illumina/Solexa 02-14-2013, 09:29 AM
Replies: 13
Views: 6,816
Posted By MLog
We tried to use the kit from the bad lot and got...

We tried to use the kit from the bad lot and got almost the same result today... The sequencing seemed to progress well until 174 cycle, but after this MCS reported an error on autofocusing ("best...
Forum: Illumina/Solexa 02-11-2013, 12:32 PM
Replies: 1
Views: 1,241
Posted By MLog
Hiseq imaging problems - dark edge

Hi everyone,

I noticed a strange effect on our HiSeq - the lower intensity of A and C on left side of the swath (attached are two thumbnails illustrationg this).
This effect is not flowcell- or...
Forum: Illumina/Solexa 01-14-2013, 12:12 PM
Replies: 55
Views: 28,203
Posted By MLog
Thanks, TonyBrooks!

Thanks, TonyBrooks!
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