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Search: Posts Made By: Markiyan
Forum: Bioinformatics 04-16-2018, 08:13 AM
Replies: 13
Views: 1,138
Posted By Markiyan
Lightbulb Use Illumina 2x250 or 2x300, not 2x150 in de novo!

Please use Illumina 2x250 or 2x300 in any de novo appication! (in addition to the pacbio).

It means 1-2 runs (2x250 or 2x300) using MiSeq or 1/2 of the HiSeq 2500 RR 2x250.

Usually it gives...
Forum: Bioinformatics 04-11-2018, 04:42 AM
Replies: 1
Views: 254
Posted By Markiyan
Lightbulb Any repeats in the loci?

Since the GATK HaplotypeCaller function involves reassembly of the reads, it may pull in the reads from the different areas for the repetitive loci.

There may be multiple causes for the problem: ...
Forum: Illumina/Solexa 04-06-2018, 02:21 AM
Replies: 13
Views: 752
Posted By Markiyan
Lightbulb rkiyan : PS: there are some GC stretches in those...

rkiyan : PS: there are some GC stretches in those reads - so please also check the thermal cycling performance of the instrument during the cluster generation.". How can I check this?.

Basically...
Forum: Illumina/Solexa 04-04-2018, 06:09 AM
Replies: 13
Views: 752
Posted By Markiyan
Exclamation PhiX% ?

1. Do you use any custom sequencing primers (esp R1)? If yes - more detail where and for which reads.

2. HOW MUCH PhiX % did you load!?? - I hope that at least 10%?

PS: there are some GC...
Forum: Illumina/Solexa 04-04-2018, 03:03 AM
Replies: 13
Views: 752
Posted By Markiyan
Lightbulb A couple things to check:

1. Did you look into
d:\Illumina\MiSeqTemp\[your run ID]\Thumbnail_Images ?
It is available only till the start of the next run.

2. Did you use PhiX in 10-15%? - at least it should show the...
Forum: Illumina/Solexa 04-04-2018, 02:06 AM
Replies: 1
Views: 378
Posted By Markiyan
First get an RFID override code...

1. you would need an RFID override code from the Illumina support.
2. The pasteur pipette should work ok (make sure it is sterile and protease/DNA'se free!)
3. If trying longer reads (>250bp), try...
Forum: Illumina/Solexa 04-04-2018, 01:59 AM
Replies: 13
Views: 752
Posted By Markiyan
Lightbulb Check sequencing adapters.

Try a new batch of the sequencing adapters.
Maybe one of your sequencing adapters had degraded?
Also check the thumbnail images from the run's tmp folder.

If both adapters at the end of the...
Forum: RNA Sequencing 03-09-2018, 08:19 AM
Replies: 2
Views: 1,126
Posted By Markiyan
Lightbulb I would go for PE reads in this case.

Assuming yours RNAseq libraries are compatible with patterned flowcells:

https://sequencing.qcfail.com/articles/?report=reader
...
Forum: Illumina/Solexa 02-23-2018, 04:45 AM
Replies: 4
Views: 1,316
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, 02:40 AM
Replies: 12
Views: 1,756
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, 09:37 AM
Replies: 5
Views: 581
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, 04:04 AM
Replies: 4
Views: 876
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, 06:33 AM
Replies: 1
Views: 726
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, 06:59 AM
Replies: 4
Views: 854
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, 09:05 AM
Replies: 10
Views: 561
Posted By Markiyan
Post Include the file name in the error message in convert2annovar.pl

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, 05:58 AM
Replies: 10
Views: 561
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:

convert2annovar.pl -format vcf4 -allsample input.vcf -outfile...
Forum: Illumina/Solexa 10-10-2017, 12:28 AM
Replies: 26
Views: 3,410
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 10-06-2017, 11:36 PM
Replies: 26
Views: 3,410
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, 04:08 AM
Replies: 1
Views: 775
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, 05:20 AM
Replies: 3
Views: 670
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, 03:12 AM
Replies: 7
Views: 715
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, 12:15 PM
Replies: 2
Views: 1,061
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, 04:56 AM
Replies: 7
Views: 981
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, 04:49 AM
Replies: 1
Views: 980
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, 05:21 AM
Replies: 5
Views: 645
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...
Showing results 1 to 25 of 109

 


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