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Forum: RNA Sequencing 11-08-2017, 07:28 AM
Replies: 5
Views: 673
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: 857
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: 857
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: 513
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: 385
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: 1,053
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: 1,053
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: 1,053
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: 310
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: 828
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: 828
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, 10:18 AM
Replies: 587
Views: 104,644
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...
Forum: Bioinformatics 05-31-2017, 10:04 AM
Replies: 587
Views: 104,644
Posted By HESmith
@jweger1988, what tools have you tried for VCF...

@jweger1988, what tools have you tried for VCF annotation and what problems have you encountered?
Forum: Bioinformatics 05-18-2017, 03:35 PM
Replies: 12
Views: 863
Posted By HESmith
IIRC, your previous posts about this topic were...

IIRC, your previous posts about this topic were deleted as inappropriate to the forum (and that hasn't changed).
Forum: Bioinformatics 03-11-2017, 04:48 PM
Replies: 2
Views: 544
Posted By HESmith
It has nothing to do with bridge amplification....

It has nothing to do with bridge amplification. WGS is whole-genome sequencing. The rRNA cluster is ~4500bp long, and a typical microbial genome is ~1000X as long. So only 1/1000 reads contain rRNA...
Forum: Pacific Biosciences 02-10-2017, 07:22 AM
Replies: 7
Views: 833
Posted By HESmith
1) What is the organism that you're studying? And...

1) What is the organism that you're studying? And are there any related genomes that might be useful for comparison?

2) Is there any possibility that the sample might be...
Forum: Bioinformatics 01-20-2017, 11:31 AM
Replies: 3
Views: 960
Posted By HESmith
There's a known incompatibility between FreeBayes...

There's a known incompatibility between FreeBayes and the CIGAR format of SAM/BAM v1.4, which results in no indel calls. If that's true for your FreeBayes VCF, reformat your alignment to v1.3 and...
Forum: Bioinformatics 01-19-2017, 12:14 PM
Replies: 7
Views: 1,100
Posted By HESmith
Paired-end reads are typically counted once...

Paired-end reads are typically counted once (unless it's the Illumina marketing team). Merged reads will also be counted once after FLASH, but unmerged reads now become two single-end reads and are...
Forum: Illumina/Solexa 01-11-2017, 12:51 PM
Replies: 2
Views: 940
Posted By HESmith
See this publication...

See this publication (https://www.ncbi.nlm.nih.gov/pubmed/?term=24886553).
Forum: Illumina/Solexa 01-10-2017, 04:21 AM
Replies: 113
Views: 18,477
Posted By HESmith
Small cores like ours appreciate the flexibility...

Small cores like ours appreciate the flexibility of the 2500, which can accommodate 16 samples per run (minimizing staff time) or two (good for uncommon sequencing requests). It's essentially a 4000...
Forum: RNA Sequencing 01-06-2017, 10:08 AM
Replies: 6
Views: 1,449
Posted By HESmith
@Shimul, it is not necessary to post the same...

@Shimul, it is not necessary to post the same question multiple times on the forum. Your run metrics look good.
Forum: Sample Prep / Library Generation 01-06-2017, 04:58 AM
Replies: 1
Views: 764
Posted By HESmith
There is no inherent limit on how much PCR...

There is no inherent limit on how much PCR product you can generate. As for any chemical reaction, the amount of product is dependent upon the amounts and concentrations of the reactants (template,...
Forum: Bioinformatics 01-05-2017, 07:53 AM
Replies: 19
Views: 2,705
Posted By HESmith
@SDos, given your concern about security, do you...

@SDos, given your concern about security, do you really expect any of us on this forum to click on blind links posted by an essentially anonymous individual?
Forum: Bioinformatics 12-06-2016, 01:39 PM
Replies: 3
Views: 817
Posted By HESmith
The bitwise flag for properly aligned pairs (0x2)...

The bitwise flag for properly aligned pairs (0x2) is set at the alignment step; that's the flag you want to use. But if you apply any other filters (such as adapter or quality trimming), then that...
Forum: Bioinformatics 11-17-2016, 12:34 PM
Replies: 7
Views: 1,020
Posted By HESmith
By conceptually similar to Moleculo, I meant that...

By conceptually similar to Moleculo, I meant that the short reads derived from a single long fragment are identified by the presence of a unique barcode/index. But I can see how this method scales...
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