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Forum: Illumina/Solexa 02-21-2019, 02:18 AM
Replies: 4
Views: 170
Posted By nucacidhunter
The calculated Tm is correct. Empirically it is...

The calculated Tm is correct. Empirically it is difficult to dissociate that oligo. For an application I tried to inhibit gap filing by both heat and a chemical method. PCR yield was decreased by 65%...
Forum: Illumina/Solexa 02-20-2019, 09:54 PM
Replies: 5
Views: 134
Posted By nucacidhunter
R1 and R2 are sequenced with different primers....

R1 and R2 are sequenced with different primers. Sequencing primers could be one of Illumina sequencing primers or custom primers based on amplicon sequence. The primer choice is based on library prep...
Forum: Illumina/Solexa 02-20-2019, 09:45 PM
Replies: 4
Views: 170
Posted By nucacidhunter
Nextera (modified Tn5 transposase) inserts ME...

Nextera (modified Tn5 transposase) inserts ME sequences by cut and paste mechanism and leaves a 9 base gap at the 3' end of insertion site. The gap is filled during 3 min incubation at 72C at PCR...
Forum: Illumina/Solexa 02-20-2019, 07:31 PM
Replies: 5
Views: 134
Posted By nucacidhunter
Paired end sequencing means that library...

Paired end sequencing means that library fragments (in this case ITS amplicons) are sequenced from both ends so R1 and R2 of the fragment are in opposite direction. If sequencing cycles are longer...
Forum: Illumina/Solexa 02-20-2019, 06:27 PM
Replies: 5
Views: 134
Posted By nucacidhunter
I assume you have done PCR wit ITS region...

I assume you have done PCR wit ITS region specific primers with no Illumina overhang or full adapter sequences. In this case they must have prepared library by adapter ligation which will result in...
Forum: Illumina/Solexa 02-19-2019, 02:19 AM
Replies: 4
Views: 133
Posted By nucacidhunter
This result could be due to following: 1-...

This result could be due to following:

1- Uneven pooling of libraries for sequencing
2- Differences in fragment size distribution of libraries (libraries with average shorter fragments will be...
Forum: RNA Sequencing 02-11-2019, 11:39 PM
Replies: 4
Views: 254
Posted By nucacidhunter
Link to Illumina Ribo-Zero Plant performance: ...

Link to Illumina Ribo-Zero Plant performance: https://www.illumina.com/products/by-type/molecular-biology-reagents/ribo-zero-rrna-removal-plant.html?langsel=/us/
Forum: RNA Sequencing 02-11-2019, 09:28 PM
Replies: 4
Views: 254
Posted By nucacidhunter
They might have used Ribo-Zero for plant...

They might have used Ribo-Zero for plant seed/root which lacks probes for removing chloroplast transcripts. This kit was discontinued late last year and updated kit is used for all plant tissue types.
Forum: Illumina/Solexa 02-07-2019, 11:27 PM
Replies: 2
Views: 288
Posted By nucacidhunter
If libraries are eluted in water, SpeedyVac is...

If libraries are eluted in water, SpeedyVac is the best option otherwise Zymo DNA Clean & Concentrator (DCC-5) which allows elution with minimum 6 ul.
Forum: Sample Prep / Library Generation 01-14-2019, 12:57 AM
Replies: 15
Views: 13,593
Posted By nucacidhunter
I can think of following two approaches: 1-...

I can think of following two approaches:

1- Their enzymatic fragmentation leaves blunt end or/and 5 overhang which can be repaired and A tailed by Taq.

2- Mix of proofreading and...
Forum: Sample Prep / Library Generation 01-03-2019, 04:11 PM
Replies: 3
Views: 601
Posted By nucacidhunter
I wonder if you are using the whole kit or just...

I wonder if you are using the whole kit or just using ligation reagents. I would not expect good ligation using only ligation reagents as this kit uses one pot workflow and skipping frag-end repair...
Forum: Sample Prep / Library Generation 12-21-2018, 04:09 PM
Replies: 15
Views: 13,593
Posted By nucacidhunter
Twist Bioscience Exome protocol does not claim...

Twist Bioscience Exome protocol does not claim end-repair at 65C. It must have been a miscommunication. Most enzymatic library prep kits include fragmentation and end repair at 20-35C and A-tailing...
Forum: Illumina/Solexa 12-18-2018, 12:03 AM
Replies: 3
Views: 528
Posted By nucacidhunter
Possible important issues: 1- degraded or...

Possible important issues:

1- degraded or low concentration of blocking oligos
2- inefficient hybridisation step (temperature, time, reaction composition)
2- non-stringent wash steps
Forum: Illumina/Solexa 12-17-2018, 11:58 PM
Replies: 2
Views: 529
Posted By nucacidhunter
High value for pre-phasing could be symptom...

High value for pre-phasing could be symptom rather than the cause. More info on phasing:
...
Forum: Illumina/Solexa 12-17-2018, 11:39 PM
Replies: 3
Views: 528
Posted By nucacidhunter
It indicates that on target reads (enrichment) is...

It indicates that on target reads (enrichment) is low and most of reads are from background. This figure for most kits is above 70%. At least one step in target capture has been non-optimal.

I...
Forum: Sample Prep / Library Generation 12-16-2018, 12:00 AM
Replies: 2
Views: 669
Posted By nucacidhunter
Double SPRI size selection window will be much...

Double SPRI size selection window will be much larger than 50 bp. For your target you need to use Sage Pippin or gel extraction.
Forum: Sample Prep / Library Generation 12-15-2018, 02:39 AM
Replies: 4
Views: 590
Posted By nucacidhunter
Bead clean up with 0.8x should remove it. It may...

Bead clean up with 0.8x should remove it. It may be necessary to do two clean ups for complete removal. Clean ups should be done with all samples that will be compared if the aim is DGE analysis.
Forum: Sample Prep / Library Generation 12-15-2018, 02:33 AM
Replies: 1
Views: 446
Posted By nucacidhunter
For PCR based amplification of 16S variable...

For PCR based amplification of 16S variable regions RNase treatment usually is not necessary. It will impact correct quantification by spectrophotometric methods if it is included in the workflow. If...
Forum: Illumina/Solexa 12-14-2018, 12:33 AM
Replies: 18
Views: 6,887
Posted By nucacidhunter
Heating denatures the oligo self-dimers and...

Heating denatures the oligo self-dimers and possible secondary structure. Skipping heating will reduce the available adapter for ligation. Its impact will depend on ratio of adapter to template RNA....
Forum: Bioinformatics 11-27-2018, 10:22 PM
Replies: 2
Views: 443
Posted By nucacidhunter
As SNPsaurus has mentioned it is more likely...

As SNPsaurus has mentioned it is more likely library prep issue. Posting FastQC output for raw data would be more informative.
Forum: Sample Prep / Library Generation 11-21-2018, 11:08 PM
Replies: 4
Views: 724
Posted By nucacidhunter
It will damage but I am not sure to what extent....

It will damage but I am not sure to what extent. You can work around it by staining and marking target sizes on the marker lane cut from the gel and using it as a guide for cutting target region from...
Forum: Sample Prep / Library Generation 11-16-2018, 10:06 PM
Replies: 5
Views: 1,231
Posted By nucacidhunter
I am not sure how you have come up with...

I am not sure how you have come up with degradation conclusion. qPCR amplicons goes through excessive cycling (normally 40) and end product can have artifacts and also running them on BA without...
Forum: Sample Prep / Library Generation 11-15-2018, 12:06 AM
Replies: 2
Views: 632
Posted By nucacidhunter
Yes it works and your plan looks fine. Second...

Yes it works and your plan looks fine. Second round of PCR adds the indexes and remaining adapter sequences and is primed from the overhang which is common among amplicons.
Forum: Bioinformatics 11-14-2018, 11:56 PM
Replies: 4
Views: 546
Posted By nucacidhunter
Stretches of G in NextSeq indicates lack of...

Stretches of G in NextSeq indicates lack of signal in those cycles and it could be result of short inserts and adapter-dimer.

In a shot gun library GC content peak will be representative of genome...
Forum: Sample Prep / Library Generation 11-14-2018, 11:46 PM
Replies: 5
Views: 1,231
Posted By nucacidhunter
Do you mean library degrades after size selection...

Do you mean library degrades after size selection and clean up if you leave the library in fridge or RT?

If the library reagents are contaminated you would not have the library in first place.
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