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Forum: Illumina/Solexa 05-25-2019, 05:51 AM
Replies: 11
Views: 1,139
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, 04:53 PM
Replies: 11
Views: 13,666
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, 04:59 AM
Replies: 2
Views: 660
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, 05:22 AM
Replies: 4
Views: 981
Posted By HESmith
Looks like adapter dimer.

Looks like adapter dimer.
Forum: Introductions 11-13-2018, 04:17 AM
Replies: 2
Views: 1,694
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, 06:08 AM
Replies: 674
Views: 167,096
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, 04:43 PM
Replies: 1
Views: 1,096
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, 09:32 AM
Replies: 2
Views: 1,205
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, 01:03 PM
Replies: 7
Views: 2,595
Posted By HESmith
https://genohub.com

https://genohub.com
Forum: Bioinformatics 03-15-2018, 06:48 AM
Replies: 674
Views: 167,096
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, 05:07 AM
Replies: 5
Views: 1,466
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, 08:34 AM
Replies: 5
Views: 1,466
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, 04:31 AM
Replies: 2
Views: 1,106
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, 06:28 AM
Replies: 5
Views: 1,926
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, 05:13 AM
Replies: 13
Views: 1,560
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, 04:14 AM
Replies: 13
Views: 1,560
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, 12:14 PM
Replies: 5
Views: 2,760
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, 07:23 AM
Replies: 1
Views: 1,063
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, 06:53 AM
Replies: 13
Views: 2,295
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, 06:15 AM
Replies: 13
Views: 2,295
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, 11:30 AM
Replies: 13
Views: 2,295
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, 11:44 AM
Replies: 3
Views: 777
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, 07:05 AM
Replies: 15
Views: 1,568
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, 06:49 AM
Replies: 15
Views: 1,568
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...
Forum: Bioinformatics 05-31-2017, 09:18 AM
Replies: 674
Views: 167,096
Posted By HESmith
Not trying to be a jerk, but have you tried...

Not trying to be a jerk, but have you tried variant calling with the test data that are included with the software? Success would eliminate installation and command errors as the source of your...
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