Question: --fr versus --rf option in Hisat2
0
gravatar for j.m.fustin
8 months ago by
j.m.fustin10
j.m.fustin10 wrote:

Hi!

We have a library of paired reads generated by the UTF-8'en-us'SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian User Manual_063017 from Takara. Read1 is the antisense strand and Read2 is the sense. I wish to use Hisat2 for alignment but I am confused by two options:

1) Specify strand information (unstranded, forward or reverse) 2) Select the upstream/downstream mate orientations for a valid paired-end alignment against the forward reference strand --fr --rf -ff

How do these 2 options relate to each other? For our library, should I select --fr or --rf? If I select --fr, should I select forward in option 1? If I select instead --rf, should I then select option 1 to reverse?

Our reads look like this: @K00339:103:HTKCFBBXX:2:1101:5436:1595 1:N:0:CTGAAGCT+GTCAGTAC AGTCGCGCTTCTTTTCCTTACTGTCTTCTTTGACAGTTTCCAGGATAAGTTTGGCCACCGAGTCATTCTTGATGTCTCTATAGTCTGTTACTGAGTCCCA + AAAF-JAFAJFFFJJJJJFJAFF-FFJJFJF<jjjjjfjjjffjjjjjjjfj<ffjfjjjjja7fjafj-<jfjfjjjjjjjjjjjjjfjj<j&lt;-ajfjf @k00339:103:htkcfbbxx:2:1101:5984:1595="" 1:n:0:ctgaagct+gtcagtac="" gagccaccttccccaccgggccttcccggccgtcccggagccggtcgcggcgcaccgccacggtggaagtgcgcccggcggcggccggtcgccggccggg="" +="" aa-fffjf-7fjjjjjjjffjjaf<jjf7fjfjjjjfjjjfjajjjjjjjjjjjjjjjjjjjjajf7jjajjjjjfjjjjjjjjjjjjjjjjfjjajffj="" @k00339:103:htkcfbbxx:2:1101:6614:1595="" 1:n:0:ctgaagct+gtcagtac="" atctggcttcctcggccccgggattcggcgaaagctgcggccggagggctgtaacactcggggtgaggtggttcggcgcgccctgagacgcgccgccccc="" +="" aaff&lt;-affjjff&lt;<jf7jj<a<f7ajfjjjfjjjjjjjf<fjafj<jjajfjffjjfjjjja7-<a-f<ff<j7fj&lt;7ajjjjff&lt;&lt;-7a<ajajj&lt;-a="" @k00339:103:htkcfbbxx:2:1101:7060:1595="" 1:n:0:ctgaagct+gtcagtac="" tggtccagaggcgcgtgtcttcctgaggctctaccagaaactgctcccgagctgtttgcaagactagttccacagaatcagtatagctaaactgaatggt="" +="" aaaffjjjjjjjjjjfjfjjfjjfjjfjjjjjjjjjjjjjjjjjfjjfjj<jjj<jjjjjfjjffjjffjjjjjjjjjjjjafjjjjjjjjjjjjjjjff="" @k00339:103:htkcfbbxx:2:1101:7527:1595="" 1:n:0:ctgaagct+gtcagtac="" gggtgcccatagaggttacaaggggccattggatccttttgacctggagttgaggaatgttagttttcaatagctgagtagtattccattgtagaaatga<="" p="">

THank you for your always excellent help!

JM

rna-seq • 448 views
ADD COMMENTlink modified 8 months ago • written 8 months ago by j.m.fustin10
0
gravatar for Jennifer Hillman Jackson
8 months ago by
United States
Jennifer Hillman Jackson25k wrote:

Hello,

See the graphic here to understand your data content: http://www.clontech.com/US/Products/cDNA_Synthesis_and_Library_Construction/Next_Gen_Sequencing_Kits/Total_RNA-Seq/xxclt_displayImage.jsp?imgCntId=136227&sitex=10020:22372:US

Your data is forward and -rf. Enter reads1 first and reads2 second on the tool form. Or if you have multiple samples, create a paired-end dataset collection with that structure.

Galaxy tutorials: https://galaxyproject.org/learn/

Thanks! Jen, Galaxy team

ADD COMMENTlink written 8 months ago by Jennifer Hillman Jackson25k
0
gravatar for j.m.fustin
8 months ago by
j.m.fustin10
j.m.fustin10 wrote:

Hi Jen,

Thanks a lot for your always useful comments. I thought it was so, but then I ran into a different problem. I first aligned with the option forward --fr, and got "70% reads aligned concordantly exactly 1 time". It seemed OK. Then, noticing my mistake with the library type, ran the same fastqsanger input files with option forward --rf and got about 98% "aligned concordantly 0 times". I still cannot explain what happened. Any idea? Both times I did input first read1 then read2 on the tool form.

It does no seem to be a QA problem because it ran OK the first time (but with the wrong option selected).

JM

ADD COMMENTlink written 8 months ago by j.m.fustin10
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 16.09
Traffic: 169 users visited in the last hour