Directional Strand related settings for RNA-seq Analysis Software
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2.6 years ago
BM ▴ 70

A question about the library preparation for RNA-seq and Directional Strand related settings for RNA-seq Analysis Software The library was prepared was using Clontech Smartseq v4 and Nextera XT So is the library directional and first strand specific (F2R1) or second strand (F1R2)? Which libray type setting should I use for HISAT2, TopHat, Feature Counts etc? Thanks in advance

RNA-Seq HiSat2 Smartseq Tophat HTseq • 937 views
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Regardless of the strandedness of the Clontech Smartseq v4 kit (which I don't know), if you then use the Nextera XT to prepare the final library, I believe it will be unstranded.

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Is this the Ultra low input kit?

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Yes it is Smartseq v4 Ultra low input kit

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2.6 years ago

Some aligners don't care about strandedness. BowTie doesn't. (Neither does STAR) FeatureCounts should have an option where you tell it if the reads should be forward or reverse. Read the documentation.

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OP is mentioning HISAT2 which does have a setting for it: --rna-strandness <string>

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2.6 years ago
Juke34 ★ 6.3k

If you want to know the type of reads you get from your library use GUESSmyLT.
When you will have your answer please let me know I would like to add it to the Library prep methods list available at the same address.

You should get a result like that:

Results of paired library inferring of reads 4_r1.sub.100000 on ref 4: 

Library type    Reads     Percent     Vizualization according to firststrand

   undecided        1        0.0%     3' -------??------- 5'
                                      5' -------??------- 3'


   ff_second        2        0.0%     3' ----------==2==> 5'
                                      5' ==1==>---------- 3'


    fr_first     4019       47.2%     3' ----------<==1== 5'
                                      5' ==2==>---------- 3'


    ff_first        5        0.1%     3' ----------<==1== 5'
                                      5' <==2==---------- 3'


   rf_second       19        0.2%     3' ----------==2==> 5'
                                      5' <==1==---------- 3'


    rf_first       21        0.2%     3' ----------==1==> 5'
                                      5' <==2==---------- 3'


   fr_second     4454       52.3%     3' ----------<==2== 5'
                                      5' ==1==>---------- 3'

Roughly 50/50 split between the strands of the same library orientation should be interpreted as unstranded.
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