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Forum: Bioinformatics 08-25-2020, 06:01 AM
Replies: 1
Views: 693
Posted By HESmith
The first error message indicates that your...

The first error message indicates that your reference genome is the culprit. Some versions contain 'N's that correspond to placeholders for the telomeric repeats. This causes reference indexing to...
Forum: Bioinformatics 08-07-2020, 11:20 AM
Replies: 2
Views: 1,063
Posted By HESmith
It's entirely normal and does not affect the...

It's entirely normal and does not affect the results.
Forum: Illumina/Solexa 08-07-2020, 05:59 AM
Replies: 3
Views: 1,045
Posted By HESmith
Yes, you can perform asymmetric runs - it's...

Yes, you can perform asymmetric runs - it's routine for single-cell RNA-Seq. Quality will decline with read length, and (in our experience) drops to unacceptable levels when you exceed the...
Forum: General 07-06-2020, 11:11 AM
Replies: 1
Views: 1,566
Posted By HESmith
Reference genomes are FASTA format. Sequencing...

Reference genomes are FASTA format. Sequencing data are FASTQ format, usually with Sanger quality scores (aka FASTQSANGER).
Forum: Bioinformatics 02-04-2020, 06:38 AM
Replies: 2
Views: 680
Posted By HESmith
1) Are you sure you have 200K-fold genome...

1) Are you sure you have 200K-fold genome coverage, or 200K reads?

2) Picard ranks duplicates by summed base-quality scores (see the documentation...
Forum: Sample Prep / Library Generation 11-07-2019, 03:51 AM
Replies: 2
Views: 1,308
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, 05:51 AM
Replies: 12
Views: 3,369
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: 15,677
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: 1,029
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: 1,932
Posted By HESmith
Looks like adapter dimer.

Looks like adapter dimer.
Forum: Introductions 11-13-2018, 04:17 AM
Replies: 2
Views: 2,002
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: 678
Views: 202,110
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: 1,577
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: 2,089
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: 5,158
Posted By HESmith
https://genohub.com

https://genohub.com
Forum: Bioinformatics 03-15-2018, 06:48 AM
Replies: 678
Views: 202,110
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: 2,296
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: 2,296
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: 1,307
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: 2,360
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: 1,979
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: 1,979
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: 8
Views: 7,404
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: 1,596
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: 3,097
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...
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