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Forum: 454 Pyrosequencing 10-12-2015, 02:44 PM
Replies: 2
Views: 4,585
Posted By martin2
Which library preparation kit did you use?...

Which library preparation kit did you use? General Library, Rapid Library, Amplicon I or II?

You mean the 4 samples were fragmeted/purified/bound to beads in large enough volumes so that you have...
Forum: 454 Pyrosequencing 07-07-2015, 01:56 PM
Replies: 3
Views: 4,932
Posted By martin2
That is expected, as I said, that is very likely...

That is expected, as I said, that is very likely the real sample barcode. Provided it is clearly present in your reads I do not see any reason why should you believe there is yet another sample...
Forum: 454 Pyrosequencing 06-29-2015, 03:08 PM
Replies: 3
Views: 4,932
Posted By martin2
The 'agagcgaa' sequence is probably a custom MID...

The 'agagcgaa' sequence is probably a custom MID (barcode). They (somebody at the sequencing centre) properly used the left trimpoint to delineate it. sff_extract does the right job as far as I can...
Forum: 454 Pyrosequencing 06-17-2015, 08:54 AM
Replies: 1
Views: 4,963
Posted By martin2
Error corrector for Illummina and Roche/454 able...

Error corrector for Illummina and Roche/454 able to also fix insertions and deletions

http://www.bioinformatics.csiro.au/blue


Below are some comments from its author from my Inbox:

<quote>...
Forum: 454 Pyrosequencing 06-17-2015, 08:42 AM
Replies: 6
Views: 5,234
Posted By martin2
You can't go back from FASTA/Q and re-create SFF....

You can't go back from FASTA/Q and re-create SFF. In theory you could but nobody wrote such code yet. As far as I am aware of the status in biopython, Roche sfftools, flower.
Forum: 454 Pyrosequencing 06-17-2015, 08:34 AM
Replies: 4
Views: 4,870
Posted By martin2
And that is exactly what ruined a number of...

And that is exactly what ruined a number of projects. I spent insane amount of time cleaning up contaminated projects. Don't do it. Although some labs could be more thorough in sonication and water...
Forum: Bioinformatics 12-19-2014, 12:31 PM
Replies: 2
Views: 2,922
Posted By martin2
I just came across this unanswered thread ... my...

I just came across this unanswered thread ... my 2c below.



First of all, not every read has an adapter, imagine a long sample insert with an adapter present way too far on the right side,...
Forum: 454 Pyrosequencing 11-11-2014, 10:29 AM
Replies: 3
Views: 1,399
Posted By martin2
Eh, I forgot that one can use the Lib-L approach...

Eh, I forgot that one can use the Lib-L approach if one uses the relevant fusion-primers, thank you for reminding me. ;-)

The emPCR kit II is a Roche term and stands for "amplicon A or paired-end"...
Forum: 454 Pyrosequencing 10-30-2014, 10:13 AM
Replies: 11
Views: 6,313
Posted By martin2
Hi, I spent 3 years analyzing public 454 data...

Hi,
I spent 3 years analyzing public 454 data and found some artefacts and have several ideas how these emerge (seems multiple mechanisms are involved). Although I haven't published the details if...
Forum: 454 Pyrosequencing 10-30-2014, 09:56 AM
Replies: 3
Views: 1,399
Posted By martin2
Hi Antony, the amplicon vs. shotgun kits use...

Hi Antony,
the amplicon vs. shotgun kits use different emPCR and hence sequencing primers. How was this ever supposed to work? You hust can't mix amplicon with shotgun samples (emPCR kit I vs kits...
Forum: De novo discovery 07-23-2014, 04:45 PM
Replies: 15
Views: 2,034
Posted By martin2
Hi pmart1, sorry I somehow did not receive an...

Hi pmart1,
sorry I somehow did not receive an email update.

The assembly you show is bad, 95% of reads having no edge means it just did not assemble, almost at all. Was this only the 454 data...
Forum: De novo discovery 06-30-2014, 03:30 PM
Replies: 15
Views: 2,034
Posted By martin2
OK, so this is likely Titanium sequencing,...

OK, so this is likely Titanium sequencing, General Library Preparation protocol or Amplicon/paired-end ..., so read length up to 500nt. The best would be to feed it into newbler:

runAssembly -o...
Forum: De novo discovery 06-30-2014, 02:57 PM
Replies: 15
Views: 2,034
Posted By martin2
sffinfo (Strain name).sff | head -n 100

sffinfo (Strain name).sff | head -n 100
Forum: De novo discovery 06-30-2014, 02:38 PM
Replies: 15
Views: 2,034
Posted By martin2
That sounds like Illumina mate-pair protocol....

That sounds like Illumina mate-pair protocol. What is the name of the file and what is the first entry or two in it?
Forum: De novo discovery 06-30-2014, 02:14 PM
Replies: 15
Views: 2,034
Posted By martin2
> ... and 250bp double end reads from a Roche 454...

> ... and 250bp double end reads from a Roche 454 GS FLX ...

What do you have? Isn't this Illumina instead?
Forum: De novo discovery 06-30-2014, 02:12 PM
Replies: 15
Views: 2,034
Posted By martin2
Hi, it happened I did all the development on my...

Hi, it happened I did all the development on my own so currently I only offer a data cleanup as a service (or even assembly). It is not only the code (28k lines of python code) but also a collection...
Forum: De novo discovery 06-30-2014, 12:50 PM
Replies: 15
Views: 2,034
Posted By martin2
Use mira assembler and make sure you feed it with...

Use mira assembler and make sure you feed it with untrimmed Illumina reads (do not do quality trimming on your own). It will remove Illumina adapters on its own. Regarding 454 data ... I cannot...
Forum: Bioinformatics 03-24-2014, 06:35 PM
Replies: 12
Views: 4,028
Posted By martin2
What is the length of the paired-end linker in...

What is the length of the paired-end linker in Illumina? 30nt or more? So in theory 22.5nt on each side? How could you map accurately 22nt piece to anything? I just don't get the idea. You just need...
Forum: RNA Sequencing 03-04-2014, 11:09 AM
Replies: 5
Views: 2,173
Posted By martin2
Provided e.g. Roche MID tags have 10 or 11 nt in...

Provided e.g. Roche MID tags have 10 or 11 nt in length, it is perfectly valid to interpret such matches are true, and trim them away with their upstream/downstream regions. But, you have to...
Forum: RNA Sequencing 03-04-2014, 06:26 AM
Replies: 4
Views: 4,784
Posted By martin2
The most important check is whether you have...

The most important check is whether you have full-length matches. Often, an N/C-terminus will be placed on a different contig/scaffold compared to the core of protein. In diploid/polypoloid organisms...
Forum: 454 Pyrosequencing 03-04-2014, 05:13 AM
Replies: 10
Views: 2,806
Posted By martin2
I developed one tool to find and remove not only...

I developed one tool to find and remove not only MIDs but also primers/adapters/artifacts from any 454 reads (works also on some IonTorrent and Illumina datasets but that's another story). I don't...
Forum: 454 Pyrosequencing 12-07-2013, 01:03 PM
Replies: 7
Views: 8,436
Posted By martin2
Hi Robin, I developed my own tool to do QC...

Hi Robin,
I developed my own tool to do QC and adapter/artifact/MID removal and I offer cleanup work as a commercial service. I think I may dare to say that I really have an overview based on more...
Forum: 454 Pyrosequencing 10-31-2013, 06:15 PM
Replies: 2
Views: 2,069
Posted By martin2
You or your technician did not install/edit the...

You or your technician did not install/edit the backup script which is ran at the end of sequencing. It is usually used to copy data from the sequencing machine (it's computer) onto a server machine....
Forum: Bioinformatics 10-31-2013, 06:04 PM
Replies: 11
Views: 6,244
Posted By martin2
+1 vote from me I would be strongly...

+1 vote from me



I would be strongly against merging SFF files together. We can be only guessing what newbler or other tools are doing while inspecting SFF data. I have a lot of experience with...
Forum: 454 Pyrosequencing 10-31-2013, 05:24 PM
Replies: 7
Views: 3,521
Posted By martin2
Rescuing the 454 run

You can disable the key check in software, it is even described in the Roche manual. Just edit the XML template file for the processing pipeline. Probably you will need to disable all filters but to...
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