<p>I received paired end amplicon sequence from a LAB with different lengths for R1 (320) and R2(280). Should I trust this lab to sequence other samples?</p>
<p>Also, I had to do a trimming over the R2 at 220(because Phred score was below 30) in the qiime2/DADA2 pipeline. Do you think trimming almost 60 bp from R2 won't result in false positives? I'm looking for precise results from my analysis. The lab claims this will give quality results as this is a specific experimental design of their lab. Should I trust the results I get from this data, also the lab? I have more samples to get sequenced.</p>

CC BY-SA 4.0
2023-07-19T15:34:11.747Z
Your title is not part of the question text; it's supposed to be a summary/primer. I'll fix it now but please do a better job next time.
Ram RS
CC BY-SA 4.0
2023-07-19T16:59:58.750Z
Please clarify your specific problem or provide additional details to highlight exactly what you need. As it's currently written, it's hard to tell exactly what you're asking.
Community
CC BY-SA 4.0
2023-07-20T05:38:21.930Z
For paired-end Amplicon sequencing the lab has provided reads with different lengths, To use it for my downstream analysis I had to trim the R2 again, making it even shorter again. I have read that trimming too much of base pairs can result in false positives, during mapping of reads to 16s Database.  So I have 2 questions, first, the above statement of false positives is right or not, Second Do you think I can use this lab for further samples as well?
abi
CC BY-SA 4.0
2023-07-21T01:22:09.740Z
There's no reason why R1 and R2 must be the same length, what did you request from the sequencing lab? Also, plenty of labs use shorter sequences for their analyses. Without knowing more about what you are trying to use these sequences for, it is impossible to answer your question.
Scot
CC BY-SA 4.0
2023-07-23T03:17:51.890Z
R1 and R2 can be different for different reasons. In your case looks like they did use different cycle of sequencing for R1 and R2. Har dot answer to your other question without knowing what the insert is or what the insert size is.
Supertech
CC BY-SA 4.0
2023-07-24T09:39:19.710Z
@Scot, I'm analyzing a person's gut microbiome. Read1 and Read2 are from the same paired-end amplicon sequencing run and originate from the same sample. Overlapping regions between Read1 and Read2 enable precise alignment and assembly, aiding identification of rare microbial species, detecting chimeric sequences, and improving alpha and beta diversity matrices. However, since R2 is shorter, I'm concerned about potential false positives in the final results due to limited overlap. Can you please advise? I'm new to Bioinformatics.
abi
CC BY-SA 4.0
2023-07-24T09:46:02.470Z
@Supertech Yes you are right, the Ct value is low for R1 and high for R2. So I guess concentration of R1 was higher than R2. Does the Ct value should remain consistent if  reads are from the same samples? Or Is it because of faults in experimental designs or condamination?
abi
CC BY-SA 4.0
2023-07-25T18:18:57.433Z
Hi @abi - I don't think anyone can tell you whether to trust your data or not - the key will be in how your analysis looks. But I can say that the trimming and the different lengths are not red flags to me. Much of the metagenomics work I've done used single end Illumina reads of 140nt, for reference.
Scot
CC BY-SA 4.0
2023-07-26T05:08:27.983Z
Thanks, @Scot That's was helpful
abi
<sequence-analysis><metagenome><illumina><trimming><microbiome>
Can files with different R1 and R2 lengths be trusted?
# Can files with different R1 and R2 lengths be trusted?

(Asked by: username_0 on 2023-07-19)

I received paired end amplicon sequence from a LAB with different lengths for R1 (320) and R2(280). Should I trust this lab to sequence other samples?

Also, I had to do a trimming over the R2 at 220(because Phred score was below 30) in the qiime2/DADA2 pipeline. Do you think trimming almost 60 bp from R2 won't result in false positives? I'm looking for precise results from my analysis. The lab claims this will give quality results as this is a specific experimental design of their lab. Should I trust the results I get from this data, also the lab? I have more samples to get sequenced.

**Tags:** sequence-analysis, metagenome, illumina, trimming, microbiome
> **Comments:**
> **username_1**: Your title is not part of the question text; it's supposed to be a summary/primer. I'll fix it now but please do a better job next time.  
> **username_2**: Please clarify your specific problem or provide additional details to highlight exactly what you need. As it's currently written, it's hard to tell exactly what you're asking.  
> **username_0**: For paired-end Amplicon sequencing the lab has provided reads with different lengths, To use it for my downstream analysis I had to trim the R2 again, making it even shorter again. I have read that trimming too much of base pairs can result in false positives, during mapping of reads to 16s Database.  So I have 2 questions, first, the above statement of false positives is right or not, Second Do you think I can use this lab for further samples as well?  
> **username_3**: There's no reason why R1 and R2 must be the same length, what did you request from the sequencing lab? Also, plenty of labs use shorter sequences for their analyses. Without knowing more about what you are trying to use these sequences for, it is impossible to answer your question.  
> **username_4**: R1 and R2 can be different for different reasons. In your case looks like they did use different cycle of sequencing for R1 and R2. Har dot answer to your other question without knowing what the insert is or what the insert size is.  
> **username_0**: @Scot, I'm analyzing a person's gut microbiome. Read1 and Read2 are from the same paired-end amplicon sequencing run and originate from the same sample. Overlapping regions between Read1 and Read2 enable precise alignment and assembly, aiding identification of rare microbial species, detecting chimeric sequences, and improving alpha and beta diversity matrices. However, since R2 is shorter, I'm concerned about potential false positives in the final results due to limited overlap. Can you please advise? I'm new to Bioinformatics.  
> **username_0**: @Supertech Yes you are right, the Ct value is low for R1 and high for R2. So I guess concentration of R1 was higher than R2. Does the Ct value should remain consistent if  reads are from the same samples? Or Is it because of faults in experimental designs or condamination?  
> **username_3**: Hi @abi - I don't think anyone can tell you whether to trust your data or not - the key will be in how your analysis looks. But I can say that the trimming and the different lengths are not red flags to me. Much of the metagenomics work I've done used single end Illumina reads of 140nt, for reference.  
> **username_0**: Thanks, @Scot That's was helpful  

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## 0 answers

