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Search: Posts Made By: nucacidhunter
Forum: Bioinformatics Yesterday, 03:14 AM
Replies: 13
Views: 1,138
Posted By nucacidhunter
This is because ONT sells their consumables ...

This is because ONT sells their consumables directly but other companies such as PacBio sell them through a local distributor that have different margin and shipping costs depending on global...
Forum: Illumina/Solexa 04-19-2018, 02:20 PM
Replies: 3
Views: 212
Posted By nucacidhunter
I should correct that by fragment numbers I meant...

I should correct that by fragment numbers I meant fragments that are flanked by both RE and are in size selection window. With a 4x6 cutter in a 30 Gb genome I guess there will a lot more restriction...
Forum: General 04-19-2018, 02:10 PM
Replies: 3
Views: 126
Posted By nucacidhunter
GBS is a common name used for even...

GBS is a common name used for even non-restriction enzyme based methods. For instance, Illumina recently released a GBS kit that prepares libraries without RE o. ddRAD generally results in good...
Forum: Illumina/Solexa 04-19-2018, 03:18 AM
Replies: 3
Views: 212
Posted By nucacidhunter
First of all standard RRBS using MspI may not be...

First of all standard RRBS using MspI may not be a good approach for plants as RRBS interrogates methylation status of C in CpG context which is most relevant for mammalians.

In a pilot RRBS you...
Forum: General 04-19-2018, 02:42 AM
Replies: 3
Views: 126
Posted By nucacidhunter
Methylation sensitive enzymes are more useful for...

Methylation sensitive enzymes are more useful for plants as their genome contains large repetitive regions due to duplication events. PstI is methylation sensitive in plants as in plants methylation...
Forum: Illumina/Solexa 04-04-2018, 01:47 PM
Replies: 13
Views: 752
Posted By nucacidhunter
Good points have been raised in posts above. A...

Good points have been raised in posts above. A run with 40% PhiX spike in should clear whether your library has any issues or if fail is due to preparation and sequencing set up.
Forum: Illumina/Solexa 04-04-2018, 03:15 AM
Replies: 13
Views: 752
Posted By nucacidhunter
if I understand correctly, individual libraries...

if I understand correctly, individual libraries BA profile was fine but the library pool did not have any DNA according to Qubit, BA and qPCR?

If library PCR amplification post adapter ligation...
Forum: Sample Prep / Library Generation 04-03-2018, 02:28 PM
Replies: 1
Views: 209
Posted By nucacidhunter
Possible causes: 1- Ligation was not successful...

Possible causes:
1- Ligation was not successful which could be due to differences in RE buffer composition not being suitable for the ligase. This could happen if restriction reaction was not...
Forum: RNA Sequencing 04-03-2018, 01:53 PM
Replies: 7
Views: 1,118
Posted By nucacidhunter
Could you also post "Per base sequence content"...

Could you also post "Per base sequence content" plot form FastQC output.
Forum: Sample Prep / Library Generation 03-31-2018, 07:54 PM
Replies: 4
Views: 675
Posted By nucacidhunter
Nextera will cut smaller DNA (I think it is above...

Nextera will cut smaller DNA (I think it is above 30 bp long) fragments as well as large ones but the chance of short fragments being cut at both ends and to be in the included size range in final...
Forum: Sample Prep / Library Generation 03-27-2018, 01:36 PM
Replies: 2
Views: 792
Posted By nucacidhunter
Counting with a hemocytometer.

Counting with a hemocytometer.
Forum: Illumina/Solexa 03-25-2018, 05:37 PM
Replies: 12
Views: 711
Posted By nucacidhunter
I assume the sequences that you have provided...

I assume the sequences that you have provided represent top strand of amplicon if put together continuously as following. ...
Forum: Illumina/Solexa 03-25-2018, 01:46 PM
Replies: 12
Views: 711
Posted By nucacidhunter
It seems that your P7 adapter is missing an A at...

It seems that your P7 adapter is missing an A at the start. The A is not part of adapter but it is added during A tailing in shotgun library prep. Since you are adding adapters as part of your GSP...
Forum: Illumina/Solexa 03-24-2018, 09:04 PM
Replies: 12
Views: 711
Posted By nucacidhunter
I wonder if you could attach the sequence of the...

I wonder if you could attach the sequence of the final amplicon with adapters that goes into sequencer. I assume you are using standard Illumina sequencing primers included in sequencing kits.
Forum: Illumina/Solexa 03-24-2018, 03:39 PM
Replies: 12
Views: 711
Posted By nucacidhunter
I do not know your library prep details but it...

I do not know your library prep details but it seems that bad amplicon R2 has not primed well possibly due to some base mismatch in adapter sequences.
Forum: Sample Prep / Library Generation 03-21-2018, 02:55 AM
Replies: 1
Views: 306
Posted By nucacidhunter
Clean up after digestion is unnecessary. If your...

Clean up after digestion is unnecessary. If your adapters disrupts the RE recognition site you just can add ligase, ATP and ligation buffer and continue with ligation. You should choose RE and...
Forum: Pacific Biosciences 03-20-2018, 12:29 AM
Replies: 6
Views: 1,090
Posted By nucacidhunter
Assembly stats is not indicator of sequence...

Assembly stats is not indicator of sequence quality as it will depend on input DNA heterozygosity, genome composition, assembly software and coverage among other factors.

Well A01 is underloaded...
Forum: Illumina/Solexa 03-16-2018, 03:25 PM
Replies: 9
Views: 1,426
Posted By nucacidhunter
A single index sequencing on HiSeq systems is...

A single index sequencing on HiSeq systems is fine and on average 98% of reads correctly demultiplex. I wonder if you have seen single index sequencing fail on MiSeq or speculating.

Edit: If PhiX...
Forum: Illumina/Solexa 03-15-2018, 01:31 PM
Replies: 9
Views: 1,426
Posted By nucacidhunter
That will work. You would have option of setting...

That will work. You would have option of setting up run with index read or without.
Forum: Bioinformatics 03-15-2018, 01:26 AM
Replies: 13
Views: 1,138
Posted By nucacidhunter
It depends on the genome. You might look at...

It depends on the genome. You might look at repeat content of a related species if it is available. I have seen Illumina based assembled contigs which were couple of hundred bases larger than the...
Forum: Illumina/Solexa 03-14-2018, 05:31 PM
Replies: 1
Views: 568
Posted By nucacidhunter
It will be useful if you can give more...

It will be useful if you can give more information on library prep method or kit to narrow on possible causes.
Forum: Core Facilities 03-14-2018, 03:00 AM
Replies: 7
Views: 997
Posted By nucacidhunter
In my experience most places: 1- pool...

In my experience most places:

1- pool multiple customers libraries only for libraries made in the facility
2- accept customer made libraries for running in a lane or flow cell in customer risk...
Forum: Bioinformatics 03-14-2018, 02:38 AM
Replies: 13
Views: 1,138
Posted By nucacidhunter
The genome size is relatively small so you will...

The genome size is relatively small so you will have better assembly if you sequence with PacBio but one SMRT cell will not be enough. You should consider 50x coverage (17.5 Gb) which will require...
Forum: Bioinformatics 03-07-2018, 10:24 PM
Replies: 8
Views: 899
Posted By nucacidhunter
Both R1s are adapter sequences (I can see the...

Both R1s are adapter sequences (I can see the index as well) suggesting that these are from adapter-dimers. After reading through adapter, polyA spacer has been sequenced and then there is no base so...
Forum: Illumina/Solexa 03-07-2018, 07:09 PM
Replies: 3
Views: 1,054
Posted By nucacidhunter
Minimum run time on Illumina systems is 4 hours...

Minimum run time on Illumina systems is 4 hours on MiniSeq and MiSeq for 36 cycles. Generally, sequencing takes long time. In every cycles depending on 1, 2 or 4 colour chemistry few steps is...
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