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Forum: Illumina/Solexa 05-17-2018, 01:56 AM
Replies: 7
Views: 605
Posted By nucacidhunter
If you are going to use commercial full-length Y...

If you are going to use commercial full-length Y adapters or synthesize oligos to make up in house adapters then you do not need to change anything. The reversing P7 and i7 index is required if you...
Forum: Epigenetics 05-16-2018, 02:33 AM
Replies: 2
Views: 142
Posted By nucacidhunter
1- Libraries has been sequenced on a NextSeq flow...

1- Libraries has been sequenced on a NextSeq flow cell that has 4 lanes so the data from lanes for each library needs to be merged into one file for each sample replicates for analysis.

2- You...
Forum: Illumina/Solexa 05-08-2018, 01:40 AM
Replies: 7
Views: 605
Posted By nucacidhunter
Following lists some standalone forked (Y)...

Following lists some standalone forked (Y) Illumina style full-length adapters:

GeneRead Adapter set A and B (12 each) from Qiagen
NEXTflex® DNA Barcodes and NEXTflex-HT Barcodes (up to 384) from...
Forum: Illumina/Solexa 05-07-2018, 03:58 AM
Replies: 7
Views: 605
Posted By nucacidhunter
You can buy adapters from several vendors...

You can buy adapters from several vendors (Illumina, Bioo Scientific, Roche and others) with up to 384 index without buying a full kit.
Forum: Illumina/Solexa 04-25-2018, 04:46 AM
Replies: 6
Views: 505
Posted By nucacidhunter
NEB recently released unique duel index primers...

NEB recently released unique duel index primers and it should be easier to use them with NEB Illumina libray prep kit.
Forum: Illumina/Solexa 04-25-2018, 04:09 AM
Replies: 6
Views: 505
Posted By nucacidhunter
There are commercially available unique duel...

There are commercially available unique duel indices from venders such as Illumina, NEB, Bioo Scientific, NuGEN (up to 96 UDI) and Integrated DNA technologies (up to 384). These are offered as...
Forum: Bioinformatics 04-22-2018, 03:14 AM
Replies: 13
Views: 1,367
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: 7
Views: 554
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: 306
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: 7
Views: 554
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: 306
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: 961
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: 961
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: 343
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: 1,339
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: 5
Views: 1,162
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: 933
Posted By nucacidhunter
Counting with a hemocytometer.

Counting with a hemocytometer.
Forum: Illumina/Solexa 03-25-2018, 05:37 PM
Replies: 12
Views: 862
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: 862
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: 862
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: 862
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: 427
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: 1,270
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: 1,497
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: 1,497
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.
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