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Forum: RNA Sequencing 09-17-2019, 06:56 PM
Replies: 2
Views: 694
Posted By nucacidhunter
RiboFree total RNA-seq library prep from Zymo...

RiboFree total RNA-seq library prep from Zymo Research looks good on paper and does not require species specific depletion step but I have not tried yet:
...
Forum: Sample Prep / Library Generation 09-14-2019, 01:24 AM
Replies: 20
Views: 6,187
Posted By nucacidhunter
Regardless of size distribution, library does not...

Regardless of size distribution, library does not seem to have typical periodicity expected in ATAC-seq libraries indicating that there was ambient DNA in nuclei stock or nuclei disintegrated during...
Forum: Ion Torrent 08-02-2019, 10:52 PM
Replies: 19
Views: 1,870
Posted By nucacidhunter
Following is the link to some OZ sequencing...

Following is the link to some OZ sequencing service providers:

http://www.agrf.org.au/contact-us

https://www.ramaciotti.unsw.edu.au/

http://dna.med.monash.edu.au/

I am sure they all will...
Forum: Ion Torrent 08-02-2019, 05:38 PM
Replies: 19
Views: 1,870
Posted By nucacidhunter
There are several companies in OZ that will...

There are several companies in OZ that will sequence your own prepared libraries. You can PM me if you one a list.
Forum: Illumina/Solexa 07-30-2019, 02:32 AM
Replies: 2
Views: 419
Posted By nucacidhunter
You can use ssDNA library prep method or a kit...

You can use ssDNA library prep method or a kit such as ACCEL-NGS 1S (or
1S Plus) DNA LIBRARY kit.
Forum: Sample Prep / Library Generation 05-25-2019, 05:48 PM
Replies: 1
Views: 723
Posted By nucacidhunter
I would suggest to do a reaction according to...

I would suggest to do a reaction according to standard protocol from start to end and check the resulting library. Tagmented DNA will not migrate according to size becuase:
1- transposon will be...
Forum: Sample Prep / Library Generation 05-23-2019, 07:25 PM
Replies: 4
Views: 765
Posted By nucacidhunter
plexWell 96 or 384 could fit your library prep....

plexWell 96 or 384 could fit your library prep. It seems to be cheap and also quick.

https://seqwell.com/product/pw96/
Forum: Illumina/Solexa 05-05-2019, 03:17 PM
Replies: 10
Views: 1,348
Posted By nucacidhunter
Any non-hot start polymerase will do the gap...

Any non-hot start polymerase will do the gap filling.
Forum: Sample Prep / Library Generation 04-05-2019, 11:09 PM
Replies: 10
Views: 1,111
Posted By nucacidhunter
Using other reverse transcriptase with strand...

Using other reverse transcriptase with strand switching activity (SMARTScribe, Maxima H Minus) might give better results. There are some threads in this site as well under single cell RNA-Seq...
Forum: Sample Prep / Library Generation 04-05-2019, 10:58 PM
Replies: 3
Views: 507
Posted By nucacidhunter
A general approach is to prime RT reaction with a...

A general approach is to prime RT reaction with a random sequences (6-8nt) followed with a defined 5' overhang and then size select the cDNA. Implementing this will depend on downstream application.
Forum: Sample Prep / Library Generation 04-05-2019, 10:45 PM
Replies: 1
Views: 535
Posted By nucacidhunter
If a library is prepared and includes a PCR step...

If a library is prepared and includes a PCR step it should sequence well regardless of input DNA quality. Following can be useful:

1- Treating input DNA with NEB FFPE repair reagents to fix some...
Forum: Illumina/Solexa 04-05-2019, 01:56 AM
Replies: 1
Views: 415
Posted By nucacidhunter
Generally smaller inserts would result in more...

Generally smaller inserts would result in more uniform coverage of genes so for counting applications short inserts are preferred and can be sequenced only 70 cycles. For other applications such as...
Forum: Sample Prep / Library Generation 04-04-2019, 03:25 AM
Replies: 4
Views: 666
Posted By nucacidhunter
Loss of large fragments mostly can be due to...

Loss of large fragments mostly can be due to inefficient binding. longer incubation of bead-DNA solution on a rotating wheel should increase recovery.
Quantifying supernatant with PicoGreen or Qubit...
Forum: Illumina/Solexa 04-04-2019, 03:12 AM
Replies: 5
Views: 865
Posted By nucacidhunter
If you do not need high number of reads, PacBio...

If you do not need high number of reads, PacBio CCS will be a good option as it will cover the whole fragment length with very high accuracy.
Forum: Bioinformatics 03-30-2019, 07:41 PM
Replies: 5
Views: 1,008
Posted By nucacidhunter
It could be result of demultiplexing HiSeq runs....

It could be result of demultiplexing HiSeq runs. i5 index read need to be masked during demultiplexing otherwise the i5 read sequences will be added to 5' end of read 2 (R4).
Forum: Illumina/Solexa 03-07-2019, 12:31 AM
Replies: 5
Views: 895
Posted By nucacidhunter
The fragment size distribution is within optimal...

The fragment size distribution is within optimal for sequence capture if that is what you are doing. To obtain larger fragments (if you are using Nextera not Nextera flex) input DNA need to be...
Forum: Illumina/Solexa 02-24-2019, 10:37 PM
Replies: 5
Views: 895
Posted By nucacidhunter
If you can post Bioanalyser result then it will...

If you can post Bioanalyser result then it will be easier to diagnose the issue.
Forum: Illumina/Solexa 02-21-2019, 01:18 AM
Replies: 10
Views: 1,348
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, 08:54 PM
Replies: 5
Views: 629
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, 08:45 PM
Replies: 10
Views: 1,348
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, 06:31 PM
Replies: 5
Views: 629
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, 05:27 PM
Replies: 5
Views: 629
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, 01:19 AM
Replies: 4
Views: 791
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, 10:39 PM
Replies: 4
Views: 794
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, 08:28 PM
Replies: 4
Views: 794
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.
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