SEQanswers

Go Back   SEQanswers > Search Forums


Showing results 1 to 25 of 500
Search took 0.02 seconds.
Search: Posts Made By: HESmith
Forum: Sample Prep / Library Generation 11-07-2019, 04:51 AM
Replies: 2
Views: 225
Posted By HESmith
The simplest way to isolate the problem is to...

The simplest way to isolate the problem is to have new facility sequence the old (high quality) sample - if the data are good, it's not the facility. Alternatively, prepare your next sample with both...
Forum: Illumina/Solexa 05-25-2019, 06:51 AM
Replies: 11
Views: 1,504
Posted By HESmith
Our small core lab likes the flexibility of the...

Our small core lab likes the flexibility of the 2500, especially Rapid mode for small numbers of samples/unusual run requests. Replacing that functionality would require two machines (although we are...
Forum: General 01-03-2019, 05:53 PM
Replies: 11
Views: 14,119
Posted By HESmith
Use BEDtools 'genomecov'...

Use BEDtools 'genomecov' (https://bedtools.readthedocs.io/en/latest/content/tools/genomecov.html) command with '-d' flag to obtain per-base depth of coverage.
Forum: Bioinformatics 01-02-2019, 05:59 AM
Replies: 2
Views: 769
Posted By HESmith
You're on the right track with BEDtools. Use the...

You're on the right track with BEDtools. Use the 'genomecov' command with the '-bg' flag (BEDGRAPH format) to calculate coverage, filter using 'awk' (awk '$4 < THRESHOLD_VALUE') to retain the...
Forum: Sample Prep / Library Generation 12-14-2018, 06:22 AM
Replies: 4
Views: 1,195
Posted By HESmith
Looks like adapter dimer.

Looks like adapter dimer.
Forum: Introductions 11-13-2018, 05:17 AM
Replies: 2
Views: 1,789
Posted By HESmith
This type of processing (parsing the barcode and...

This type of processing (parsing the barcode and UMI) is standard for scRNA-Seq data. Would you post the header and first 10 lines of the BAM file? That would help us to troubleshoot your problem.
Forum: Bioinformatics 08-09-2018, 07:08 AM
Replies: 674
Views: 174,960
Posted By HESmith
@JenBarb see this thread...

@JenBarb see this thread (https://www.biostars.org/p/297273/#297277) in Biostars.
Forum: Sample Prep / Library Generation 07-30-2018, 05:43 PM
Replies: 1
Views: 1,218
Posted By HESmith
Yes, adapter dimers can anneal to...

Yes, adapter dimers can anneal to insert-containing molecules via the adapter ends. The simplest solution is to perform one new round of PCR using ~1/2 (or less, depending upon the amount) of the...
Forum: Bioinformatics 07-24-2018, 10:32 AM
Replies: 2
Views: 1,390
Posted By HESmith
Explanation here...

Explanation here (https://academic.oup.com/bioinformatics/article/29/4/444/200320) (see section 2.3).
Forum: Service Providers 04-04-2018, 02:03 PM
Replies: 7
Views: 3,077
Posted By HESmith
https://genohub.com

https://genohub.com
Forum: Bioinformatics 03-15-2018, 07:48 AM
Replies: 674
Views: 174,960
Posted By HESmith
You can try the reformat.sh command per @GenoMax...

You can try the reformat.sh command per @GenoMax but with 'sam=1.3' flag. That worked for me with a different variant caller (FreeBayes).
Forum: Bioinformatics 02-28-2018, 06:07 AM
Replies: 5
Views: 1,652
Posted By HESmith
From the manual...

From the manual (https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/dedupe-guide/): "Pair Limitations: Dedupe supports paired reads, but it was not really designed for them. When...
Forum: Bioinformatics 02-27-2018, 09:34 AM
Replies: 5
Views: 1,652
Posted By HESmith
Remove the spaces after the equals (=) signs and...

Remove the spaces after the equals (=) signs and try again.
Forum: Sample Prep / Library Generation 02-02-2018, 05:31 AM
Replies: 2
Views: 1,146
Posted By HESmith
Expression profiles can change in minutes, so I'd...

Expression profiles can change in minutes, so I'd recommend adding a stabilization component (e.g., Qiagen's RNAprotect) immediately after collection. At that point, the samples would be stable for...
Forum: RNA Sequencing 11-08-2017, 07:28 AM
Replies: 5
Views: 2,002
Posted By HESmith
The band is adaptor dimers. They form clusters....

The band is adaptor dimers. They form clusters. Use gel or bead purification to remove.
Forum: Illumina/Solexa 11-01-2017, 06:13 AM
Replies: 13
Views: 1,677
Posted By HESmith
Short templates/adapter dimers produce a drop-off...

Short templates/adapter dimers produce a drop-off in signal intensity, which @ATGCT doesn't observe (first thumbnail). And adapter dimers produce a diagnostic spiky signal, also not observed.
Forum: Illumina/Solexa 10-31-2017, 05:14 AM
Replies: 13
Views: 1,677
Posted By HESmith
The run is significantly over-clustered, as...

The run is significantly over-clustered, as indicated by the low PF (only 68%; should be mid-80s to mid-90s) in combination with the high error rate (4.4%; should be <1%). Quantify your libraries by...
Forum: Illumina/Solexa 09-26-2017, 01:14 PM
Replies: 5
Views: 3,137
Posted By HESmith
Read 1 begins with the RT primer. It contains the...

Read 1 begins with the RT primer. It contains the cell barcode, UMI, and oligo(dT), which primes 1st strand synthesis from the mRNA's poly(A) tail. Until you get past the poly(A), which can be long,...
Forum: Bioinformatics 07-31-2017, 08:23 AM
Replies: 1
Views: 1,157
Posted By HESmith
Adapter ligation is a concentration-dependent...

Adapter ligation is a concentration-dependent reaction. If enrichment is performed first, then the amount of one of the reactants (fragments) becomes low, which reduces the efficiency of the reaction...
Forum: Illumina/Solexa 07-27-2017, 07:53 AM
Replies: 13
Views: 2,459
Posted By HESmith
Yes, the longer oligos are more expensive. But...

Yes, the longer oligos are more expensive. But with dual indexing, 20 primers can accommodate 100 samples.

One more point to consider: PCR-free ligation prep requires a substantial amount (1-2...
Forum: Illumina/Solexa 07-27-2017, 07:15 AM
Replies: 13
Views: 2,459
Posted By HESmith
Ligation is always less efficient than...

Ligation is always less efficient than incorporating the adapter ends directly into your amplicon primers. Is there a reason NOT to use the following type of primer?

adapter end - index - random...
Forum: Illumina/Solexa 07-26-2017, 12:30 PM
Replies: 13
Views: 2,459
Posted By HESmith
I was wondering how you'd generate amplicons w/o...

I was wondering how you'd generate amplicons w/o PCR, but see that one round is acceptable. You can incorporate the sequencing adaptor ends into your target sequence primers, with random k-mers...
Forum: Bioinformatics 06-14-2017, 12:44 PM
Replies: 3
Views: 827
Posted By HESmith
It's not actually VCard format. It's just that...

It's not actually VCard format. It's just that VCard uses the same file extension (.vcf) as VCF, and your computer is configured to read the former by default. Open the VCF in a flat text editor and...
Forum: Bioinformatics 06-07-2017, 08:05 AM
Replies: 15
Views: 1,655
Posted By HESmith
Try BBMap's 'covstats' command - I think it...

Try BBMap's 'covstats' command - I think it provides a summary of coverage.
Forum: Bioinformatics 06-07-2017, 07:49 AM
Replies: 15
Views: 1,655
Posted By HESmith
You can determine how much of your genome is...

You can determine how much of your genome is covered, and at what read depth, using BEDTools 'genomecov' command (see here (http://bedtools.readthedocs.io/en/latest/content/tools/genomecov.html) for...
Showing results 1 to 25 of 500

 


All times are GMT -8. The time now is 10:20 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO