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Forum: Illumina/Solexa 02-23-2018, 05:45 AM
Replies: 4
Views: 919
Posted By Markiyan
Exclamation 600 cycle miseq asymetric runs & seq alternatives for 600bp+ amplicons.

I had tried R1:319 I1:6 R2:300 and I must admit that as you approach 300 mark the quality decay (error rates increase) is quite exponential in it's nature :-(, so the reported quality at [email protected] was...
Forum: Illumina/Solexa 02-21-2018, 03:40 AM
Replies: 10
Views: 1,070
Posted By Markiyan
Lightbulb Try having a look at thumbnails or raw images.

In this case first try having a look at the raw thumbnail/image data, and make a movie from the run's tiles (using imagemagic, one frame per cycle).

This can help a lot in understanding what...
Forum: Bioinformatics 11-30-2017, 10:37 AM
Replies: 5
Views: 535
Posted By Markiyan
Lightbulb After 8 theads the speedup for cufflinks/diff is marginal...

From my experience the speed up of the cufflinks/cuffdiff is marginal after 8 threads...

In some cases the runtime with 32-48 threads may be way longer than with 8-16, esp on systems with 4+ CPU...
Forum: Pacific Biosciences 11-22-2017, 05:04 AM
Replies: 4
Views: 717
Posted By Markiyan
Lightbulb Use multipass pacbio reads for self error correction and Kmer counting.

First try filtering out the multipass reads, and using those for kmer counting and self error correction.

Make sure to remove any mitochondrial/symbionts reads before doing the kmer counting....
Forum: General 11-06-2017, 07:33 AM
Replies: 1
Views: 547
Posted By Markiyan
Lightbulb Some insetrions are flanked with duplication or deletion events...

Quite a few insertional events (esp transposase induced are flanked by the target sequence duplication).
In other cases it can be accompanied by the region deletion/replacement, so it would have...
Forum: Pacific Biosciences 10-30-2017, 07:59 AM
Replies: 4
Views: 696
Posted By Markiyan
Lightbulb The pacbio library & sequencing artefacts are the main cause of trouble.

The frequency of the pacbio Pacbio library & sequencing artefacts: chimeras (2%-10%) and siameras (1%-3%) would be 3-5 orders of magnitude higher than genuine meiosis recombination events (every...
Forum: Bioinformatics 10-25-2017, 10:05 AM
Replies: 10
Views: 515
Posted By Markiyan
Post Include the file name in the error message in

We need to improve our exception handling a bit.
We should always include the name on the file which our program tries to open for writing in the error message. Also differentiate between file open...
Forum: Bioinformatics 10-25-2017, 06:58 AM
Replies: 10
Views: 515
Posted By Markiyan
Exclamation Do not use stdin or stdout words for regular filenames.

The stdout and stdin are reserved filenames in the unix/perl world...

So avoid using them in regular file names.

Better do: -format vcf4 -allsample input.vcf -outfile...
Forum: Illumina/Solexa 10-10-2017, 01:28 AM
Replies: 26
Views: 2,983
Posted By Markiyan
Lightbulb Inline indexes & patterned flowcells.

In that case we need to have first 4-5 bases as a random sequence (for clusters ID), followed by the barcode (another 4-8 bases), than the insert...

This is really important for RNAseq & CHIPseq...
Forum: Illumina/Solexa 10-07-2017, 12:36 AM
Replies: 26
Views: 2,983
Posted By Markiyan
Lightbulb We need to move index closer (start) of the sequencing read... - Like 454-MID.

Since index hopping seems to be caused by recombinase, recombining over a homologus sequencing/index primer binding sequence, what about using 454-MID style indexing - devote first 6-8 basepairs of...
Forum: Pacific Biosciences 09-20-2017, 05:08 AM
Replies: 1
Views: 628
Posted By Markiyan
Lightbulb Make sure to have Illumina data from the same DNA prep for efficient error-correction

Two points:

1. To get more DNA try making the sample prep more efficient/use more starting material/

2. To allow efficient error-correction of the pacbio reads also prep PCR-free (optional: +...
Forum: Illumina/Solexa 08-23-2017, 06:20 AM
Replies: 3
Views: 641
Posted By Markiyan
Lightbulb Is the machine on a service contract? (In...

Is the machine on a service contract?

(In case it is mechnical failure):
If yes, than ask the Illumina rep to regrease with a suitable lubricant ALL moving parts that require lubrication.
Forum: Bioinformatics 08-23-2017, 04:12 AM
Replies: 7
Views: 672
Posted By Markiyan
Lightbulb Some commandline examples for mapping quality filtering.

Mapping score distribution depends on mapper program/version used.

First check the actual MAPQ score histogram for your bam file (first 1M of reads):

samtools view $1 | head - -n 1000000 | cut...
Forum: Bioinformatics 08-14-2017, 01:15 PM
Replies: 2
Views: 873
Posted By Markiyan
Lightbulb If you want predictable fastq-dump performance......

If you want predictable fastq-dump performance... use local data sources.

1. Forget about the wi-fi. Use only wired network (suitable docking station with ethernet if on macbook).

2. Download...
Forum: Sample Prep / Library Generation 07-28-2017, 05:56 AM
Replies: 7
Views: 936
Posted By Markiyan
Exclamation Any residual DNA-ase in CHIP sample?

Is there any residual DNA-ase left in the CHIP seq DNA sample, after the DNA digestion?

If yes, it can chew back the adapter ends, allowing some of them to ligate into the dimers...
Forum: Ion Torrent 07-26-2017, 05:49 AM
Replies: 1
Views: 809
Posted By Markiyan
Lightbulb First have a look at your library prep. Than on processing.

First it would be help full if you could provide a bit more info about the sample/library prep used.

Actually adapter artefacts can come in many variations, esp if you have a bit of nuclease...
Forum: Illumina/Solexa 07-13-2017, 06:21 AM
Replies: 5
Views: 591
Posted By Markiyan
Lightbulb Try assembling less data first... Use MiSeq 2x250 or 2x300...

First I would try assembling less data, and see what are the most abundant species in the datasets... Than filter it out and repeat with more data...

Also I would use 4 channel Illumina sequences...
Forum: Bioinformatics 05-23-2017, 10:20 AM
Replies: 8
Views: 1,095
Posted By Markiyan
Lightbulb For NAS-es, you can go with high end Qnap's or Thecuses:

I've got Thecus N10850, for 1.8K, popped in 32GB of RAM (4x8GB DDR3L 1600) instead of 4GB stock, and than created raid 6 on 9x6TB WD-red pro drives. You can also use 8TB HE filled drives, just DON'T...
Forum: General 05-19-2017, 03:47 AM
Replies: 14
Views: 1,877
Posted By Markiyan
It is a desktop with 1TB HDD (SSD)...

I would like to see a core i5 desktop supporting 1TB of RAM....

I think here a user has 4GB - 32GB of RAM and 1TB of HDD/SSD...
Forum: Bioinformatics 04-18-2017, 06:28 AM
Replies: 0
Views: 478
Posted By Markiyan
Chimera reads detection in the WGS dataset

Are there any stand alone tools for chimeric reads/clones detection in the de novo WGS datasets - like cosmid/BAC clones, sanger reads, moleculo mini asms or pacbio/nanopore error corrected...
Forum: RNA Sequencing 04-06-2017, 03:28 AM
Replies: 1
Views: 722
Posted By Markiyan
Lightbulb Search for experimental and analysis protocols used to generate the data.

So first try to find the detailed description of their experimental and data analysis protocols and pipelines used to generate the results you've downloaded.

Also try downloading some aml RNAseq...
Forum: Bioinformatics 04-06-2017, 03:19 AM
Replies: 2
Views: 796
Posted By Markiyan
Lightbulb Find the detailed description of their RNAseq experimental & data analysis protocol.

Search for some recent papers on amyloid leukemia, where they had submitted the RNAseq data to the SRA and have a look at NCBI's SRA and EBI's ENA.
Forum: General 03-28-2017, 09:26 AM
Replies: 2
Views: 910
Posted By Markiyan
Lightbulb You probably want to use miSeq in 2x250 for data aquisition.

A couple of points with respect of experimental design:

1. Make sure the Illumina libraries are PCR-free prep.
2. Unless you want to do 96 genomes on one hiseq lane, use MiSeq, since hiseq would...
Forum: Illumina/Solexa 03-14-2017, 03:59 AM
Replies: 2
Views: 856
Posted By Markiyan
Lightbulb Looks like there is a problem with imaging one of the flowcell surfaces...

It looks like there is a problem with imaging one of the flowcell surfaces...

Either focusing issue (Z axis lens mover in a need of some cleaning/re greasing/calibration) or lots of very small...
Forum: Bioinformatics 03-13-2017, 04:16 AM
Replies: 8
Views: 2,436
Posted By Markiyan
Lightbulb Try removing rRNA, tRNA, transposon reads.

In order to minimise the impact of the bug, try removing the reads matching rRNA, tRNA and transposase genes present in a genome of the analysed organism and rerunning the mapping/analysis.

Showing results 1 to 25 of 101


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