Quality Score

Question: Quality Score

7.3 years ago by

Peng, Tao • 170

Peng, Tao • 170 wrote:

Hi jen, I followed the GALAXY web cast to check the quality of RNA-seq data: one sample seem to have score above 20 in most bases (R2); but the other one is around 6-8 in most bases (R4) (see the attached PDF files). Does this mean R4 RNA-seq data are BAD? What exactly does it mean anyway? Thanks for your help, tao To: galaxy-user@bx.psu.edu Cc: Peng, Tao Subject: visualization of alignment Hello Tao, For the Bowtie results, the aligned results may be low because the data is RNA and not DNA. TopHat is generally considered a better choice for RNA since it allows for bridges over splice sites (introns). The full documentation for each program is on each tool's form and/or you can contact the tool authors with scientific questions at tophat.cufflinks@gmail.com. Also, a tutorial and FAQ are available here: http://usegalaxy.org/u/jeremy/p/galaxy-rna-seq-analysis-exercise http://usegalaxy.org/u/jeremy/p/transcriptome-analysis-faq For visualization, an update that allows the use of a user-specified fasta reference genome is coming out very soon. For now, you can view annotation by creating a custom genome build, but the actual reference will be not included. Use "Visualization -> New Track Browser" and follow the instructions for "Is the build not listed here? Add a Custom Build". Help for using the tool is available here: http://galaxyproject.org/Learn/Visualization As stated before, please email the mailing list directly and not individual team members. Specifically, with a "to" to the mailing list (only) and not including team members as a "to" or "cc" unless ask to do so when sharing private data. Our internal tracking system and public archives rely on this method. Thank you for your future corporation. Best, Jen Galaxy team -- Jennifer Jackson http://usegalaxy.org http://galaxyproject.org/Support

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modified 7.3 years ago • written 7.3 years ago by Peng, Tao • 170

7.3 years ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

===> Please use "Reply All" when responding to this email! <=== Hi jen, I followed the GALAXY web cast to check the quality of RNA-seq data: one sample seem to have score above 20 in most bases (R2); but the other one is around 6-8 in most bases (R4) (see the attached PDF files). Does this mean R4 RNA-seq data are BAD? What exactly does it mean anyway? Thanks for your help, tao To: galaxy-user@bx.psu.edu Cc: Peng, Tao Subject: visualization of alignment Hello Tao, For the Bowtie results, the aligned results may be low because the data is RNA and not DNA. TopHat is generally considered a better choice for RNA since it allows for bridges over splice sites (introns). The full documentation for each program is on each tool's form and/or you can contact the tool authors with scientific questions at tophat.cufflinks@gmail.com. Also, a tutorial and FAQ are available here: http://usegalaxy.org/u/jeremy/p/galaxy-rna-seq-analysis-exercise http://usegalaxy.org/u/jeremy/p/transcriptome-analysis-faq For visualization, an update that allows the use of a user-specified fasta reference genome is coming out very soon. For now, you can view annotation by creating a custom genome build, but the actual reference will be not included. Use "Visualization -> New Track Browser" and follow the instructions for "Is the build not listed here? Add a Custom Build". Help for using the tool is available here: http://galaxyproject.org/Learn/Visualization As stated before, please email the mailing list directly and not individual team members. Specifically, with a "to" to the mailing list (only) and not including team members as a "to" or "cc" unless ask to do so when sharing private data. Our internal tracking system and public archives rely on this method. Thank you for your future corporation. Best, Jen Galaxy team -- Jennifer Jackson http://usegalaxy.org http://galaxyproject.org/Support

ADD COMMENT • link written 7.3 years ago by Jennifer Hillman Jackson ♦ 25k

===> Please use "Reply All" when responding to this email!<=== Hello Tao, The tool "NGS: QC and manipulation -> FastQC" (last tool in group) may be helpful for your project. In general, sequence with quality scores this low would be considered unusable. Perhaps double check the options used with the Fastq Groomer tool? Or check/filter the data before grooming? This may not be the case for your data, but just in case, please note that CASAVA 1.8+ now produces both filtered and unfiltered results and would need to be used with the "Sanger" option with the "Fastq Groomer" tool. This prior Q&A explains the filtering: http://gmod.827538.n3.nabble.com/Filtering-Illumina-CASAVA-1-8-FASTQ- files-tt3233562.html Hopefully this helps. Please send future questions directly to the mailing list as the "to" recipient. There is no need to send directly "to" or as "cc" any of the Galaxy team directly. This helps us to track and address questions quickly and as a team. Best, Jen Galaxy team -- Jennifer Jackson http://usegalaxy.org http://galaxyproject.org/Support

ADD REPLY • link written 7.3 years ago by Jennifer Hillman Jackson ♦ 25k

7.3 years ago by

Peng, Tao • 170

Peng, Tao • 170 wrote:

Thanks, jen. I have asked informatic scientists at Hutch to do the QC for me and both R2 and R4 are ok from FASTQC analysis. My question is: Do I still need to use the groomer in GALAXY and use the groomed data for further analysis such as TOPHAT? Should I skip the steps to compute quality statistics and draw boxplots using the groomed data? Thanks, tao To: galaxy-user Cc: Peng, Tao Subject: Re: [galaxy-user] quality score ===> Please use "Reply All" when responding to this email!<=== Hello Tao, The tool "NGS: QC and manipulation -> FastQC" (last tool in group) may be helpful for your project. In general, sequence with quality scores this low would be considered unusable. Perhaps double check the options used with the Fastq Groomer tool? Or check/filter the data before grooming? This may not be the case for your data, but just in case, please note that CASAVA 1.8+ now produces both filtered and unfiltered results and would need to be used with the "Sanger" option with the "Fastq Groomer" tool. This prior Q&A explains the filtering: http://gmod.827538.n3.nabble.com/Filtering-Illumina-CASAVA-1-8-FASTQ- files-tt3233562.html Hopefully this helps. Please send future questions directly to the mailing list as the "to" recipient. There is no need to send directly "to" or as "cc" any of the Galaxy team directly. This helps us to track and address questions quickly and as a team. Best, Jen Galaxy team -- Jennifer Jackson http://usegalaxy.org http://galaxyproject.org/Support

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