Closed ZiggeyQi closed 6 months ago
Hi Ziggey - so you are not using any 3'end protocols but you do full -length transcript sequencing?
Yes, i plan to use the SLAM-seq sample to construct the standarded strand-specific library to get the transcribed RNA strand info. There is no 3'-polyA enrichment, just following the instruction of commercial strand-specific library construction kit, the typical strand-specific library construction main steps including: rRNA removement, fragmentation, reverse transcription, NGS-sequencing(PE150, not full length). All the things were same as the SLAM-seq protocol, just replace the “QuantSeq 3′mRNA-Seq Library Prep Kit” as “KAPA Stranded RNA-Seq Library Preparation Kit” to get the strand-specific library. Most RNA types and immature mRNAs will unbiasedly contained in the strand-specific library, how can i use slamdunk help me to get the T>C conversion and unlabeled reads under this context.
The SLAMDUNK developer (who initially responded to your question) will likely have more to say, but I will offer these two comments:
Best, Isaac
Hi, Isaac, thanks a lot for your helpful comments. So i can use HISAT-3N to align my trimmed reads to reference genome, then using the BAM file as the input of SLAMDUNK to do the next steps, just minding the tags generated by HISAT-3N matchs to the NextGenMap's format, also, i can just use the pipeline bam2bakR to get my expected outputs.
Hi @ZiggeyQi and @isaacvock
first of all thanks @isaacvock for the comprehensive answer, I second all what you are saying.
As a third option, I could also refer you directly to the Ameres lab that developed SLAMseq, because afaik they have also a full-length SLAMseq pipeline set up (I vaguely recall STAR + custom quantification library), I could double check with them and put you in contact (or add them here) if you are interested @ZiggeyQi ?
Best,
Tobi
Hi, Tobi, thank you for providing me the opportunity to contact Ameres lab, now the standarded SLAMseq with fine-tuning the library construction and analysis steps already mets my research goals. Thanks again for your helpful reply @t-neumann , @isaacvock
Hi @ZiggeyQi - in case you want to reach out to the contact - Niko Popitsch - I tagged him here so you can follow up @popitsch
OK, thanks a lot.
Hi there, if i wanna use 4sU pulsed total RNA to construct strand-specific library followed by the SLAM-seq protocol with the exception of library construction step, in this context, rRNA will be removed but no polyA enrichment steps for library construction. With this strategy, can slamdunk helpful to identify the new synthetized RNA for further analysis without the 3'-UTR annotation information provided. I expected to get the strand informations of both nascent and steady RNAs, is there any advices for me to achieve my goal. Your reply is highly anticipated.
Best, Ziggey