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Forum: Illumina/Solexa 02-23-2018, 04:29 PM
Replies: 2
Views: 140
Posted By nucacidhunter
1 ng DNA of the organism will have 23,200-46,300...

1 ng DNA of the organism will have 23,200-46,300 genome equivalents. Assuming 1Kb average fragment size and 25% conversion rate 1 ng DNA will result in 231M unique fragments. If you sequence the...
Forum: Sample Prep / Library Generation 02-22-2018, 06:04 PM
Replies: 5
Views: 3,236
Posted By nucacidhunter
AMPure PB are specific formulation of AMPure XP...

AMPure PB are specific formulation of AMPure XP for PacBio library clean ups and costs more than XP.
Forum: Illumina/Solexa 02-21-2018, 03:26 PM
Replies: 3
Views: 214
Posted By nucacidhunter
It is possible that in your cells accessible...

It is possible that in your cells accessible chromatin region frequency is low so less DNA is available for tagmentation.

Library profile does not look like ATAC-Seq as it lacks periodicity....
Forum: Illumina/Solexa 02-21-2018, 01:28 PM
Replies: 3
Views: 214
Posted By nucacidhunter
One cause could be low number of tagmented...

One cause could be low number of tagmented amplifiable fragments (fragmenst that have different tags in each end and are in a size range that PCR settings allows amplification). I wonder if you have...
Forum: De novo discovery 02-09-2018, 05:22 PM
Replies: 2
Views: 415
Posted By nucacidhunter
You already have a good N50 with long reads. The...

You already have a good N50 with long reads. The options are combination of 10x Link reads, Hi-C and optical mapping. Searching " Vertebrate Genomes Project Plans to Combine Technologies for 'Near...
Forum: Illumina/Solexa 02-09-2018, 05:06 PM
Replies: 6
Views: 865
Posted By nucacidhunter
My comment is regarding sequencing metrics. I...

My comment is regarding sequencing metrics. I have not followed up assembly stats.
Forum: Illumina/Solexa 02-09-2018, 02:17 AM
Replies: 4
Views: 410
Posted By nucacidhunter
You library should sequence on MiSeq and there is...

You library should sequence on MiSeq and there is no need for custom sequencing primers.
Forum: Illumina/Solexa 02-08-2018, 02:49 PM
Replies: 6
Views: 865
Posted By nucacidhunter
Nextera works well up to 75% GC and does not...

Nextera works well up to 75% GC and does not require spike in for increasing sequence diversity.
Forum: Illumina/Solexa 02-08-2018, 02:43 PM
Replies: 4
Views: 410
Posted By nucacidhunter
Illumina sequencing primer mixes are proprietary....

Illumina sequencing primer mixes are proprietary.
If those are full length sequences of your adapter oligos, your library fragments will not cluster as they are missing flow cell binding motives...
Forum: Illumina/Solexa 02-02-2018, 09:05 PM
Replies: 6
Views: 865
Posted By nucacidhunter
These two issues can have common or separate...

These two issues can have common or separate causes. To diagnose the issue it will be helpful if you can post run summary page (Reads tables) and also %Base from data by cycle plot of analysis page...
Forum: Illumina/Solexa 01-30-2018, 05:30 PM
Replies: 1
Views: 366
Posted By nucacidhunter
You can multiplex up to 24 samples/libraries...

You can multiplex up to 24 samples/libraries provided that each has different index. For low pooling (less than 6) you need pay attention to index sequences to maximize base diversity...
Forum: Illumina/Solexa 01-29-2018, 09:03 PM
Replies: 2
Views: 609
Posted By nucacidhunter
This depend on qPCR cycle settings. Using...

This depend on qPCR cycle settings. Using standard protocols gives enough time for extension of around up to 900 bp fragments so larger fragments are not amplified by qPCR. Since fragments above this...
Forum: Illumina/Solexa 01-25-2018, 01:15 AM
Replies: 23
Views: 3,207
Posted By nucacidhunter
Your statement is correct but UDI addresses...

Your statement is correct but UDI addresses another issue (index hopping) in patterned flow cells used in HiSeq 3000/4000 HiSeqX and NovaSeq to maintain fidelity of reads assigned to multiplexed...
Forum: Illumina/Solexa 01-17-2018, 08:09 PM
Replies: 23
Views: 3,207
Posted By nucacidhunter
Unknown reads source in indexed sequencing: ...

Unknown reads source in indexed sequencing:

1- PhiX
2- indices that does not match those in sample sheet caused by base mis-match in excess of set numbers in bcl2 fastq. This is result of poor...
Forum: Sample Prep / Library Generation 01-17-2018, 08:02 PM
Replies: 6
Views: 4,531
Posted By nucacidhunter
Yes. Bead solution to DNA solution volume ratio.

Yes. Bead solution to DNA solution volume ratio.
Forum: Illumina/Solexa 01-17-2018, 03:18 PM
Replies: 23
Views: 3,207
Posted By nucacidhunter
I am just reading between the lines and assume...

I am just reading between the lines and assume that a pool of library was sequenced on both machines and a particular library yielded more reads on HiSeq than MiSeq. Some possible explanations:
...
Forum: Illumina/Solexa 01-17-2018, 01:33 PM
Replies: 23
Views: 3,207
Posted By nucacidhunter
They work fine on NovaSeq and HS4000 and should...

They work fine on NovaSeq and HS4000 and should be fine on MiSeq too as Innovelty has mentioned.

I wonder if you have used adapters directly from IDT or Illumina branded plates from Illumina. It...
Forum: Illumina/Solexa 01-10-2018, 02:42 AM
Replies: 14
Views: 1,994
Posted By nucacidhunter
You can have either desalting or gel...

You can have either desalting or gel purification. The trade off is between cost of purification and potentially loosing reads because of short and incomplete or oligos with deletion that will affect...
Forum: Illumina/Solexa 01-09-2018, 08:00 PM
Replies: 14
Views: 1,994
Posted By nucacidhunter
You can add barcodes to primers in your case if ...

You can add barcodes to primers in your case if you amplify large targets with few overlapping amplicons.

For barcoded adapters you can use the Illumina adapter oligos and add the designed...
Forum: Illumina/Solexa 01-09-2018, 05:14 AM
Replies: 14
Views: 1,994
Posted By nucacidhunter
You can add inline barcode to the 5' end of...

You can add inline barcode to the 5' end of primer(s) to generate barcoded amplicons. If the amplicon targets limited number of region resulting in low diversity then you can have variable length...
Forum: Illumina/Solexa 01-03-2018, 01:35 PM
Replies: 2
Views: 447
Posted By nucacidhunter
A stretch of A homopolymer after reading through...

A stretch of A homopolymer after reading through adapters is from immobilized oligo C and D on flow cell but it should appear at the 3' end of sequence. In your example it is at 5' end. Look at post...
Forum: Sample Prep / Library Generation 12-21-2017, 03:14 PM
Replies: 8
Views: 625
Posted By nucacidhunter
It does not seem to be result of...

It does not seem to be result of over-amplification. Most likely cassette lane has been faulty or other weak cause could be amplicon contamination from the lab if you have recently processed...
Forum: Sample Prep / Library Generation 12-21-2017, 02:33 PM
Replies: 8
Views: 625
Posted By nucacidhunter
So you have used in total 20 ng of size selected...

So you have used in total 20 ng of size selected tags for PCRs and got 45.6 ng/ul after pooled PCR clean up. I am interested to know what was the elution volume from beads to look at possibility of...
Forum: Sample Prep / Library Generation 12-21-2017, 01:57 PM
Replies: 8
Views: 625
Posted By nucacidhunter
If you have run size selected tags on BA or still...

If you have run size selected tags on BA or still have remaining to run you can check for the presence of those peaks. I think the size selection has been sub optimal.

Other weak possibility is...
Forum: Illumina/Solexa 12-20-2017, 08:28 PM
Replies: 4
Views: 726
Posted By nucacidhunter
I am pointing to index diversity not the library....

I am pointing to index diversity not the library. I wonder which wells from adapter plate you have used.
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