Question: Directional Strand related settings for RNA-seq Analysis Software
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gravatar for BM
6 weeks ago by
BM40
United Kingdom
BM40 wrote:

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

ADD COMMENTlink modified 6 weeks ago by Juke-342.2k • written 6 weeks ago by BM40

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.

ADD REPLYlink written 6 weeks ago by h.mon25k

Is this the Ultra low input kit?

ADD REPLYlink written 6 weeks ago by genomax67k

Yes it is Smartseq v4 Ultra low input kit

ADD REPLYlink written 6 weeks ago by BM40
0
gravatar for swbarnes2
6 weeks ago by
swbarnes25.5k
United States
swbarnes25.5k wrote:

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.

ADD COMMENTlink modified 6 weeks ago • written 6 weeks ago by swbarnes25.5k

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

ADD REPLYlink written 6 weeks ago by genomax67k
0
gravatar for Juke-34
6 weeks ago by
Juke-342.2k
Sweden
Juke-342.2k wrote:

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.
ADD COMMENTlink modified 6 weeks ago • written 6 weeks ago by Juke-342.2k
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