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Forum: Oxford Nanopore 09-29-2020, 06:05 AM
Replies: 11
Views: 10,608
Posted By Markiyan
Instead of covaris G-tube use syringe with needle for framentation.

Unfortunately covaris G-tubes have too small opening for getting DNA bigger than <10kbp, and fast flow generates a lot of shotr fragments.
for hitting 20-49kbp fargments range I recommend using a 5...
Forum: Bioinformatics 06-21-2018, 12:55 AM
Replies: 1
Views: 1,043
Posted By Markiyan
Lightbulb You can do custom trimming (based on different...

You can do custom trimming (based on different profile of errors between R1&R2 (to take into account the higher chance of presence of the erroneous/random data in the end of the R2 read)).

Forum: Illumina/Solexa 06-13-2018, 02:14 AM
Replies: 5
Views: 2,053
Posted By Markiyan
Novaseq ws Nextseq 500?

Dear GenoMax,

How does the quality (raw error rates) of the 2x150 runs compare with nextseq 500 2x150?

Are they quite similar given the 2 channel imaging?
And averaging 0.5%-2% errors per...
Forum: Bioinformatics 06-11-2018, 08:47 AM
Replies: 9
Views: 4,720
Posted By Markiyan
A couple of points: For taxonomy - specific...

A couple of points:

For taxonomy - specific blast it is way better to create a subset(s) of the blast db in the fasta format and format them as a separate databases... - it would help reduce IO...
Forum: Bioinformatics 06-08-2018, 05:05 AM
Replies: 9
Views: 4,720
Posted By Markiyan
1. Make sure the blast volume #43 is not...

1. Make sure the blast volume #43 is not corrupted, check it's md5 sums.
2. Ensure that your systems has enough RAM available to blast process.
3. Please put your blast db on the DAS (Direct...
Forum: Bioinformatics 05-17-2018, 01:08 AM
Replies: 4
Views: 3,337
Posted By Markiyan
Lightbulb RDBMS are usually more efficient with these types of queries...

It looks like it tries to ungzip and scan whole input file for each chromosome postion range...
So to do it 68 thousand times... it takes a bit of time...

to fix:
1. Your perl script need the...
Forum: Bioinformatics 05-11-2018, 04:19 AM
Replies: 6
Views: 2,446
Posted By Markiyan
Lightbulb Also you can use blast or mummer to detect the overlapping ends...

First you need to detect by how much the ends are overlapping.
Than you can save non-overlapping portion + a single copy of the overlapping area sequence to a file.

You can detect overlapping...
Forum: Bioinformatics 04-16-2018, 08:13 AM
Replies: 13
Views: 2,881
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: 1,783
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: 14
Views: 4,231
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?.

Forum: Illumina/Solexa 04-04-2018, 06:09 AM
Replies: 14
Views: 4,231
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: 14
Views: 4,231
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: 1,460
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: 14
Views: 4,231
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: 3,660
Posted By Markiyan
Lightbulb I would go for PE reads in this case.

Assuming yours RNAseq libraries are compatible with patterned flowcells:
Forum: Illumina/Solexa 02-23-2018, 04:45 AM
Replies: 4
Views: 3,925
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 R1@319bp was...
Forum: Illumina/Solexa 02-21-2018, 02:40 AM
Replies: 12
Views: 3,963
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: 1,688
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: 3,747
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: 1,547
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, 06:59 AM
Replies: 4
Views: 3,135
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: 1,924
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, 05:58 AM
Replies: 10
Views: 1,924
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, 12:28 AM
Replies: 26
Views: 13,459
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: 13,459
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...
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