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Forum: Sample Prep / Library Generation 08-30-2018, 09:32 AM
Replies: 6
Views: 473
Posted By M4TTN
We were using NexteraXT for another project up...

We were using NexteraXT for another project up until our stocks ran out. We've not tried Flex yet. Same cost though I think.

I'm not sure AmpliSeq would work for our question. We only have a...
Forum: Sample Prep / Library Generation 08-30-2018, 02:12 AM
Replies: 6
Views: 473
Posted By M4TTN
Cost-effective sequencing of >200 20kb amplicons

Dear all, title says it all:

We are looking into the best/most efficient/cost effective way of fragmenting/barcoding/sequencing a ~20kb target/fragment from ~200 independent sources. Final read...
Forum: Illumina/Solexa 12-14-2017, 04:45 AM
Replies: 7
Views: 1,263
Posted By M4TTN
Fair comment. Certainly not wanting to upset the...

Fair comment. Certainly not wanting to upset the machine. I did see a user on here extend the sequencing run on a MiSeq reagent kit and got more than an extra 50 cycles.

We are currently...
Forum: Illumina/Solexa 12-14-2017, 01:16 AM
Replies: 7
Views: 1,263
Posted By M4TTN
2x42bp + 6bp index worked perfectly. Approx 190...

2x42bp + 6bp index worked perfectly. Approx 190 K/mm clusters, and >95% PF, yielded a total of 480M paired reads. Q-scores look excellent.

I am tempted to try incrementing the read length in...
Forum: Illumina/Solexa 12-13-2017, 06:15 AM
Replies: 7
Views: 1,263
Posted By M4TTN
Thanks for the link: it looks like 2x42 + 6 = 90...

Thanks for the link: it looks like 2x42 + 6 = 90 is within range (91 cycles).
I'll report back if there is a problem.
Forum: Illumina/Solexa 12-13-2017, 02:26 AM
Replies: 7
Views: 1,263
Posted By M4TTN
Maximum read cycles in NextSeq kits?

Hello,
We are using the 75 cycle 400M kit for paired-end sequencing.
Our understanding is that there are 75 read cycles, plus another 16 cycles (2x8 cycles) available for barcode detection. Total...
Forum: Sample Prep / Library Generation 08-09-2016, 08:54 AM
Replies: 18
Views: 3,656
Posted By M4TTN
Dear NextGenSeq, (apologies for dragging up an...

Dear NextGenSeq, (apologies for dragging up an old post!)

I have a DNA enrichment project where our inserts range from only 40bp and up. We'd like to get them all.

We've just done a run and it...
Forum: Sample Prep / Library Generation 03-31-2016, 07:15 AM
Replies: 48
Views: 24,591
Posted By M4TTN
Hi anamar, I am pretty sure that all the...

Hi anamar,

I am pretty sure that all the Bioanalyser lanes converge into one area on the chip - at the point that they are "analysed". Each sample reaches there at different times based on the...
Forum: Illumina/Solexa 01-27-2016, 08:47 AM
Replies: 31
Views: 9,211
Posted By M4TTN
Depends on how many reads you need...we are...

Depends on how many reads you need...we are multiplexing 5-8 samples quite nicely on a MiSeq (counting expt). No need to go to higher depth for us (at least not at the moment).
Forum: Illumina/Solexa 01-27-2016, 08:04 AM
Replies: 31
Views: 9,211
Posted By M4TTN
Info from Illumina: The MiniSeq is similar...

Info from Illumina:

The MiniSeq is similar to the technology utilised with our NextSeq platform based on 2-channel SBS chemistry.

Pricing Information

MiniSeq System (SY-420-1001): 35,653...
Forum: Illumina/Solexa 10-14-2015, 08:14 AM
Replies: 6
Views: 1,776
Posted By M4TTN
Indeed, these are different samples each time. ...

Indeed, these are different samples each time.

Any idea what might have caused the first library to be so much better?

We prepare genomic DNA by a similar method each time and prepare samples...
Forum: Illumina/Solexa 10-14-2015, 07:47 AM
Replies: 6
Views: 1,776
Posted By M4TTN
Yes, we've contact tech support. They responded...

Yes, we've contact tech support. They responded last week, but nothing since...

The run is definitely below spec. When using v3 chemistry, the published specs state 2x300 should generate %Q30 >70%...
Forum: Illumina/Solexa 10-13-2015, 11:34 PM
Replies: 6
Views: 1,776
Posted By M4TTN
Here are the FWHM plots: ...

Here are the FWHM plots:

https://www.dropbox.com/s/d0k73ecj0mpfh61/Run12%20FWHM.pdf?dl=0

The bottom surface is broader and spikier - but not sure if this is a significant (meaningful)...
Forum: Illumina/Solexa 10-07-2015, 08:31 AM
Replies: 6
Views: 1,776
Posted By M4TTN
MiSeq lower quality on the bottom surface

Hello all,

Just wondering if anyone else that uses a MiSeq has observed this issue? We invariably see that the error rate and quality scores are worse on the bottom flow cell surface than the top....
Forum: Bioinformatics 04-08-2015, 06:51 AM
Replies: 63
Views: 6,252
Posted By M4TTN
Actually this is a very good point that I knew...

Actually this is a very good point that I knew about (from reading your post earlier) but recently forgot about. It turns out that part of the poor mapping of the recent run (aside from issues with...
Forum: Bioinformatics 04-02-2015, 12:57 AM
Replies: 63
Views: 6,252
Posted By M4TTN
Data in all cases is whole genome sequencing of...

Data in all cases is whole genome sequencing of S.cerevisiae genomic DNA (also containing mitochondiral DNA and an endogenous plasmid element). Prepared using NexteraXT using all standard conditions...
Forum: Bioinformatics 04-01-2015, 07:30 AM
Replies: 63
Views: 6,252
Posted By M4TTN
Hi Genomax. Actually we are really quite...

Hi Genomax. Actually we are really quite perplexed, and are starting to wonder if our last run was particularly poor quality (for as yet unknown reasons).

On the first run (3 months ago; 2x300),...
Forum: Bioinformatics 04-01-2015, 05:43 AM
Replies: 63
Views: 6,252
Posted By M4TTN
Actually I am not sure that quality drops off...

Actually I am not sure that quality drops off much at all. It appears to in the MiSeq reporter software, but this is just because so many inserts are shorter than the read length - and as Brian...
Forum: Bioinformatics 04-01-2015, 02:56 AM
Replies: 63
Views: 6,252
Posted By M4TTN
It turns out that on the last run (350+250),...

It turns out that on the last run (350+250), adapter trimming had not been enabled in MISeq. We are requeuing the analysis with Illumina so at least the output fastas will have been processed the...
Forum: Bioinformatics 03-31-2015, 02:09 PM
Replies: 63
Views: 6,252
Posted By M4TTN
As far as I know we do just run a FASTQ only on...

As far as I know we do just run a FASTQ only on the MiSeq. But my understanding was that the output sequences are already pretrimmed.

Thanks for the bbduk options explanation - I am slowly getting...
Forum: Bioinformatics 03-31-2015, 01:11 PM
Replies: 63
Views: 6,252
Posted By M4TTN
The files are correctly paired since the...

The files are correctly paired since the resulting sam files we generate clearly have pairs of reads in sequential lines that map to the same genomic location (and most of the time overlap...
Forum: Bioinformatics 03-31-2015, 12:21 PM
Replies: 63
Views: 6,252
Posted By M4TTN
Thanks Brian. I need to check when back in work...

Thanks Brian. I need to check when back in work tomorrow, but I don't think I used fasta files that had been processed with bbduk first for this bbmap attempt. I think they were the raw files...
Forum: Bioinformatics 03-31-2015, 04:03 AM
Replies: 63
Views: 6,252
Posted By M4TTN
bbmap is taking quite a long time... (8 core mac...

bbmap is taking quite a long time... (8 core mac mini).

In the absence of a growing output file, is there any way to tell how far it may have got? It's been running for at least 15 mins now at...
Forum: Bioinformatics 03-31-2015, 04:02 AM
Replies: 63
Views: 6,252
Posted By M4TTN
We are making progress: At least in some of the...

We are making progress: At least in some of the samples, the 10% unmapped reads are coming from the 2 micron plasmid (approx 200x copies per cell it seems), and also from mitochondrial genome, which...
Forum: Bioinformatics 03-27-2015, 08:14 AM
Replies: 63
Views: 6,252
Posted By M4TTN
Hmmm.. a quick manual BLAST alignment of half a...

Hmmm.. a quick manual BLAST alignment of half a dozen of the unmapped sequences spat out by SamTools reveals that they map to the yeast 2 micron plasmid!

If this is representative sample, it...
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