bbmerge high percentage of no solution reads
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11 weeks ago
eli_bayat ▴ 80

Hello, I have a paired end reads and I am running bbmerge to merge the reads into a fastq file. However, in log file, I see high percentage of no solution reads.

Pairs:                  681070
Joined:                 262235          38.503%
Ambiguous:              15798           2.320%
No Solution:            403037          59.177%
Too Short:              0               0.000%
Adapters Expected:      798584          58.627%
Adapters Found:         6               0.000%

Avg Insert:             213.9
Standard Deviation:     20.3
Mode:                   222

Insert range:           35 - 294
90th percentile:        222
75th percentile:        222
50th percentile:        222
25th percentile:        221

I have two questions and appreciate any help:

  1. Any idea what might be the cause of this high percentage of no solution reads?
  2. What my options are to find the reason for this?

To find the root cause the only thing I can think of is to map the unpaired reads (those did not merge) to the reference and look at the alignment or try to manually pair them.

Thanks

no_solution BBmerge sequencing merg • 375 views
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What is the average read length? Why are you trying to merge the R1 and R2 reads?

Keep in mind that BBmerge works only if the reads R1 and R2 overlaps

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thanks for the response, reads are 2x150 bp and amplicon size is 230-ish. reads are being merged to then be matched a reference.

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Unless you have explicitly designed your libraries such that (number_of_sequencing_cycles) > (insert_size /2), the reads are not going to overlap.

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Thanks for the response, the number of sequencing cycles is > insert size/2.

One thing I noticed by looking into fastqc results is that the adapters started quite early on into the reads, I wonder if this indicates a sequencing or amplification issue. I see in bbmerge output adapters expected is very high as well. Could this be the issue? Could you please explain what does adapter expected/found means and when they are high, what it means? enter image description here

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You have inserts that are shorter than the expected size. So this would indicate some issue with amplification (if you were expecting only full length products).

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