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Forum: RNA Sequencing 01-14-2020, 06:31 AM
Replies: 1
Views: 573
Posted By Michael.Ante
For solid tissue analysis, you have the...

For solid tissue analysis, you have the assumption, that your observed xNA (miroRNA, RNA, DNA, etc.) is derived from one origin showing a certain 'fingerprint'. Meaning that your observation is...
Forum: Sample Prep / Library Generation 10-25-2019, 02:56 AM
Replies: 1
Views: 977
Posted By Michael.Ante
Hi, What library prep have you used? Do...

Hi,

What library prep have you used?
Do you differentiate between MT-rRNA and nucleic rRNA? The mitochondrial rRNA molecules have a polyA tail and are therefore detectable after polyA selection....
Forum: Bioinformatics 10-25-2017, 12:41 AM
Replies: 2
Views: 1,468
Posted By Michael.Ante
Hi DMNO, did you take the transcripts'...

Hi DMNO,

did you take the transcripts' strand-orientation into account? Try using RSeQC's infer-experiment tool (http://rseqc.sourceforge.net/#infer-experiment-py). It gives you a good way to...
Forum: Bioinformatics 02-21-2017, 12:36 AM
Replies: 1
Views: 1,980
Posted By Michael.Ante
I don't know if it's a good idea to do it this...

I don't know if it's a good idea to do it this way, since you'll get a higher amount of overlapping features. I hope you have a stranded library.

Nevertheless, in order to get what you described...
Forum: RNA Sequencing 02-16-2017, 01:03 AM
Replies: 4
Views: 1,520
Posted By Michael.Ante
Hi Danielle, I hope it's not to late for a...

Hi Danielle,

I hope it's not to late for a suggestion. The Genecode and ENSEMBL annotations include in their gtf files per gene a field called "gene_biotype".
If you use htseq-count (for...
Forum: Bioinformatics 01-24-2017, 01:00 AM
Replies: 2
Views: 1,586
Posted By Michael.Ante
Hi bbm, I think the problem is not the name...

Hi bbm,

I think the problem is not the name field, but the missing information on exon-intron structure (since the function "convertBed2Introns" called the error).
Parsing a GFF3 file into...
Forum: Bioinformatics 01-11-2017, 01:50 AM
Replies: 1
Views: 1,188
Posted By Michael.Ante
Hi Marcos, It seems that your plant is a...

Hi Marcos,

It seems that your plant is a model species and well annotated. You could download the gene annotation and extract the 5' and 3' UTR from the gff3 file.
In order to incorporate the...
Forum: Bioinformatics 12-20-2016, 02:07 PM
Replies: 9
Views: 3,362
Posted By Michael.Ante
It should be something like awk...

It should be something like
awk 'NR%4==2{print}' in.fastq | wc
With the awk command, you print the nucleotides, with wc you count the output's characters.
Forum: Bioinformatics 12-19-2016, 08:45 AM
Replies: 16
Views: 4,679
Posted By Michael.Ante
Did you see this pattern also with the...

Did you see this pattern also with the "--nogroup" option?
The bases are binned without that option; which let the distribution may look smoother than it is. The last base, shown in your figure, is...
Forum: RNA Sequencing 12-15-2016, 04:57 AM
Replies: 18
Views: 3,457
Posted By Michael.Ante
Hi chiayi, in your BBmap index, the naming...

Hi chiayi,

in your BBmap index, the naming of the chromosome is "Chr2 CHROMOSOME dumped from ADB: Jun/20/09 14:54; last updated: 2009-02-02", whilst in the Hisat alignment it's just "Chr2". Either...
Forum: Bioinformatics 11-29-2016, 03:21 AM
Replies: 5
Views: 4,955
Posted By Michael.Ante
Try as a first solution: countdata <-...

Try as a first solution:
countdata <- as.matrix(read_excel("DEseqcounts.xlsx"),header=TRUE, row.names=1)

And check then
summary(is.numeric(countdata[,1]))

Maybe there are some empty lines...
Forum: Bioinformatics 11-28-2016, 11:51 PM
Replies: 5
Views: 4,955
Posted By Michael.Ante
I'm not so familiar with the stringtie pipeline,...

I'm not so familiar with the stringtie pipeline, but I recommend avoiding Excel for most NGS related analyses (see Zeeberg et al. 2004: Mistaken Identifiers: Gene name errors can be introduced...
Forum: RNA Sequencing 10-06-2016, 01:24 AM
Replies: 5
Views: 1,547
Posted By Michael.Ante
Hi, Before alignment I'd perform QC with...

Hi,

Before alignment I'd perform QC with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)and FastQC screen (http://www.bioinformatics.babraham.ac.uk/projects/fastq_screen/)...
Forum: Bioinformatics 09-22-2016, 02:13 AM
Replies: 5
Views: 2,614
Posted By Michael.Ante
Maybe you have also the wrong file: vs.

Maybe you have also the wrong file:


vs.
Forum: Bioinformatics 09-22-2016, 01:37 AM
Replies: 5
Views: 2,614
Posted By Michael.Ante
Hi, did you got any error messages, while...

Hi,

did you got any error messages, while doing:

?
You forgot to include the header which should lead to a non-functional bam file.
Please try samtools view -bSh output_E1.sam >...
Forum: Bioinformatics 09-20-2016, 03:45 AM
Replies: 4
Views: 1,397
Posted By Michael.Ante
The bam_stat.py was a suggestion since it also...

The bam_stat.py was a suggestion since it also works for DNA-seq alignments (you'll hopefully don't see spliced reads).
You can also have a look at the QC-metrics from Picard tools, or have a look...
Forum: Bioinformatics 09-20-2016, 01:19 AM
Replies: 4
Views: 1,397
Posted By Michael.Ante
Hi Rangika, 2nd first: You need to be...

Hi Rangika,

2nd first:
You need to be aware of the fact that samtools flagstat produces statistics on alignments. Meaning, a read can align multiple time and will occur multiple times in the...
Forum: Bioinformatics 09-07-2016, 01:00 AM
Replies: 3
Views: 1,497
Posted By Michael.Ante
I'm sure bbmap...

I'm sure bbmap (http://seqanswers.com/forums/showthread.php?t=41057) can do that.
STAR works very well with Illumina reads and the guys from Pacific Bioscience did some parameter optimisation to...
Forum: Bioinformatics 09-06-2016, 07:48 AM
Replies: 15
Views: 2,884
Posted By Michael.Ante
Not in the first 6 month; that the usual...

Not in the first 6 month; that the usual probation period. In this period you can be fired without reasons, but you can also just leave.
After this period it is clearly more difficult to fire...
Forum: RNA Sequencing 08-25-2016, 11:41 PM
Replies: 6
Views: 1,684
Posted By Michael.Ante
Hi Sebastian, the latest versions of STAR...

Hi Sebastian,
the latest versions of STAR report the NH field. In older versions you should be able to report the number of hits with an according parameters setting.
Forum: RNA Sequencing 08-23-2016, 01:14 AM
Replies: 6
Views: 1,684
Posted By Michael.Ante
Hi Sebastian, You should have a look at the...

Hi Sebastian,

You should have a look at the rRNA content. Download just the sheep's rRNA sequences from NCBI as fasta, create a bowtie2 index, and map the reads with bowtie2 against them. The...
Forum: RNA Sequencing 05-24-2016, 11:47 PM
Replies: 6
Views: 3,212
Posted By Michael.Ante
Hi Ivygreen, did you receive the same map...

Hi Ivygreen,

did you receive the same map mass as with cuffdiff? If not, you can use e.g. R to analyse the genes.fpkm_tracking and isoforms.fpkm_tracking files. For instance, you can make a...
Forum: RNA Sequencing 05-24-2016, 08:32 AM
Replies: 1
Views: 1,876
Posted By Michael.Ante
Hi meabh, First, the main difference is that...

Hi meabh,

First, the main difference is that TopHat2 is designed to detect and report spliced reads. Whenever there is s a read spanning two exons, you'll see that with TopHat2 alignment. The...
Forum: Bioinformatics 05-18-2016, 04:41 AM
Replies: 2
Views: 1,541
Posted By Michael.Ante
Hi Heso, You can use RSeQC's bam2fq.py...

Hi Heso,

You can use RSeQC's bam2fq.py (http://rseqc.sourceforge.net/#bam2fq-py) to make a fastq file out of your "out_no_feature.sam".
In order to get the header into your files, use: samtools...
Forum: Bioinformatics 05-13-2016, 05:22 AM
Replies: 4
Views: 1,445
Posted By Michael.Ante
Hi, A) and B) are different releases of the...

Hi,

A) and B) are different releases of the Human gene assembly. So don't mix them.
If you want to have a bowtie index and its corresponding transcriptome index, download the Fasta files and the...
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