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Forum: RNA Sequencing 05-28-2014, 01:30 PM
Replies: 8
Views: 2,767
Posted By zzhao2
Downloaded. Thank you!

Downloaded. Thank you!
Forum: RNA Sequencing 05-28-2014, 01:05 PM
Replies: 8
Views: 2,767
Posted By zzhao2
Hi Brian, I just tried the link of repair.sh but...

Hi Brian, I just tried the link of repair.sh but it was BBMap, and I didn't seem to see that file in the source file list?
Forum: RNA Sequencing 05-28-2014, 01:00 PM
Replies: 8
Views: 2,767
Posted By zzhao2
Thank you so much! I'll try them.

Thank you so much! I'll try them.
Forum: RNA Sequencing 05-28-2014, 12:30 PM
Replies: 8
Views: 2,767
Posted By zzhao2
Thanks for your reply. I didn't merge two sets of...

Thanks for your reply. I didn't merge two sets of mapped data. I used cutadapt separately for left and right read files to trim off some adapter sequences, low quality bases, and too-short reads,...
Forum: RNA Sequencing 05-28-2014, 08:55 AM
Replies: 8
Views: 2,767
Posted By zzhao2
TopHat, trimmed PE reads, and SAM flags

Hi All,
I used cutadapt to trim my PE reads and TopHat to map the trimmed ones. TopHat could map most of them. Then I wanted to use HTSeq-Count to count the reads. However it complained like this:...
Forum: Bioinformatics 02-14-2014, 10:40 AM
Replies: 22
Views: 4,034
Posted By zzhao2
Just FYI, I tried trimmomatic with different...

Just FYI, I tried trimmomatic with different palindrome clip thresholds including 10,15,20, and 30, and all gave me very similar numbers of dropped sequences. I think this is consistent with the...
Forum: Bioinformatics 02-14-2014, 09:00 AM
Replies: 22
Views: 4,034
Posted By zzhao2
Here are the plots with the --nogroup option that...

Here are the plots with the --nogroup option that I forgot to attach.
Forum: Bioinformatics 02-14-2014, 08:56 AM
Replies: 22
Views: 4,034
Posted By zzhao2
Do you mean that it's safe to remove duplicates...

Do you mean that it's safe to remove duplicates marked by Picard? I think it's easier to check PCR duplicates for PE reads. What about SE reads? Is Picard equally reliable or not?
Forum: Bioinformatics 02-14-2014, 08:52 AM
Replies: 22
Views: 4,034
Posted By zzhao2
I've run FastQC with --nogroup. This is very...

I've run FastQC with --nogroup. This is very helpful. I see 5 or 6 bases with abnormal GC. Please see attached plots.

I used the Illumina's TruSeq3-PE-2.fa file provided by trimmomatic. The...
Forum: Bioinformatics 02-14-2014, 08:05 AM
Replies: 22
Views: 4,034
Posted By zzhao2
What I got from the core facility is the separate...

What I got from the core facility is the separate fastq files, a R1 and a R2 file per sample. So I assume that the samples have been demultiplexed. If in case the tags are left in the sequences, is...
Forum: Bioinformatics 02-14-2014, 07:42 AM
Replies: 22
Views: 4,034
Posted By zzhao2
My two colleagues did their RNA-Seq experiments...

My two colleagues did their RNA-Seq experiments at different times, and they study different problems. One had 6 samples sequenced maybe 6 months ago (let me call it exp1), and the other had 18...
Forum: Bioinformatics 02-14-2014, 06:56 AM
Replies: 22
Views: 4,034
Posted By zzhao2
Agree. And as I have mentioned, I have two...

Agree. And as I have mentioned, I have two independent sets of data and both have similar FastQC plots. Maybe our core facility consistently has some problem. I'll check with them.

Thank you all...
Forum: Bioinformatics 02-13-2014, 01:19 PM
Replies: 22
Views: 4,034
Posted By zzhao2
Well, I've used trimmomatic to get rid of...

Well, I've used trimmomatic to get rid of adapters, N's and low quality bases for the sample shown in those plots in my initial post. I used the example parameter settings in the trimmomatic's manual...
Forum: Bioinformatics 02-13-2014, 07:54 AM
Replies: 22
Views: 4,034
Posted By zzhao2
I tried the Q5 trimming using FastX with a...

I tried the Q5 trimming using FastX with a typical sample, but no reads were trimmed. I think it's because all bases in my reads have a score higher than 5. So why did I get those Ns at the end of...
Forum: Bioinformatics 02-12-2014, 09:42 AM
Replies: 22
Views: 4,034
Posted By zzhao2
HI jwfoley, Thank you so much for the...

HI jwfoley,
Thank you so much for the informative reply. So it sounds like I can do a Q5 trimming as well as removing adapters. I don't know the details about library preparation, so I'll ask my...
Forum: Bioinformatics 02-12-2014, 08:40 AM
Replies: 22
Views: 4,034
Posted By zzhao2
Trim/filter or not?

Hello,
I have some paired-end RNA-Seq reads (length=100) sequenced using Illumina's HiSeq platform and was wondering whether I should trim/ilter my reads. I've run FastQC with some of my samples and...
Forum: Bioinformatics 01-27-2014, 12:01 PM
Replies: 1
Views: 1,952
Posted By zzhao2
Please ignore my initial post. The real reason...

Please ignore my initial post. The real reason why my read is not mapped is not the "N", it's because in the FLAG field it's marked as "0x10 SEQ being reverse complemented". When I change this single...
Forum: Bioinformatics 01-27-2014, 11:37 AM
Replies: 1
Views: 1,952
Posted By zzhao2
htseq-count: reads with letter "N"

Hi,
I've just found that a read starting with letter "N" in my sam file was marked as "no_feature" by htseq-count. I hacked that read by changing the "N" to the reference letter and also modifying...
Forum: Bioinformatics 01-16-2014, 08:05 AM
Replies: 5
Views: 5,296
Posted By zzhao2
Hi kmcarr, Thanks for pointing this out. I...

Hi kmcarr,
Thanks for pointing this out. I didn't specify the stranded option because my reads are stranded. I've actually tried all three settings: yes, no, and reverse, but none of them gave a...
Forum: Bioinformatics 01-16-2014, 07:57 AM
Replies: 5
Views: 5,296
Posted By zzhao2
Hi Dario, thanks for your reply. Yes I've...

Hi Dario,
thanks for your reply. Yes I've noticed the chromosome name issue and changed them to chr1, chr2, etc. Without changing them I could only get a result with all genes' read counts being...
Forum: Bioinformatics 01-14-2014, 11:03 AM
Replies: 5
Views: 5,296
Posted By zzhao2
htseq-count: many reads are "no_feature".

Hi,
I just started to use htseq-count for my paired-end RNA-Seq data. I've name-sorted uniquely mapped reads outputted by tophat, converted the sorted bam file to sam, and used the sam file with an...
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