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yes. It did work.. I then split the fastq files per barcode using fastx barcode splitter. However, it still did not solve my problem of less number of reads being aligned after running tophat. Also, fastq files I obtained from fastx and casava were totally different!
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How did you run casava? What is your input for fastx barcode splitter and how did you start it? And, what is "different"? What did you expect?Originally posted by biofreak View Post
SvenComment
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well my barcodes are not illumina but are nugen. I ran casava normally with the added
use-bases-mask parameter. it did not complain and generated fastq files. When I ran tophat with these files, it somehow could not align most of the reads. Final read count of SAM files was in thousands or even less in some cases.
I then generated 1 fastq files per lane through casava ignoring the barcodes. Then used barcodespliiter to split the fastq file according to the barcode.
For any sample, fastq file generated this way did not match with the one generated by casava. (in terms of number of lines as well as contents).
Also, tophat alignment does better job then the previous version. But the line counts of the SAM file are still not in millions.. I am not sure of my results at this point.Comment
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I ran barsplitter as follows:
cat combined.fastq | fastx_barcode_splitter.pl --bcfile ../barcode1.txt --bol --mismatches 1 --prefix "lane1" --suffix ".fastq"
It creates separate fastq files but barcodes are retained in the file. So I removed those (first 4) first using:
fastx_trimmer -i fastqfile -o trim_fastqfile -f 5 -l 50 -Q 33
then ran tophat on the fastq files.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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