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Bowtie alignment

I got the flagstat result from my Bam file, while the mapping rate is high, the percentages of properly paired reads and singletons are relatively low, what should I have to do?

112103606 + 0 in total (QC-passed reads + QC-failed reads)
112103606 + 0 primary
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
0 + 0 primary duplicates
112103606 + 0 mapped (100.00% : N/A)
112103606 + 0 primary mapped (100.00% : N/A)
112102614 + 0 paired in sequencing
56339631 + 0 read1
55762983 + 0 read2
2720182 + 0 properly paired (2.43% : N/A)
109362326 + 0 with itself and mate mapped
2740288 + 0 singletons (2.44% : N/A)
101228694 + 0 with mate mapped to a different chr
88514230 + 0 with mate mapped to a different chr (mapQ>=5)

EDIT:

I used bcl2fastq with this command and adapter removing

bcl2fastq -R /proj/naiss2023-22-1174/Masomeh/240131_NB551428_0057_AHT2V3BGXV \
    --input-dir /proj/naiss2023-22-1174/Masomeh/240131_NB551428_0057_AHT2V3BGXV/Data/Intensities/BaseCalls/ \
    --output-dir /proj/naiss2023-22-1174/Masomeh \
    --interop-dir /proj/naiss2023-22-1174/Masomeh/240131_NB551428_0057_AHT2V3BGXV/Data/Intensities/InterOp/ \
    --stats-dir /proj/naiss2023-22-1174/Masomeh/240131_NB551428_0057_AHT2V3BGXV/Data/Intensities/BaseCalls/Stats/ \
    --reports-dir /crex/proj/naiss2023-22-1174/Masomeh/BaseCalls/Reports/ \
    --sample-sheet 'ATACsamplesheet.csv' \
-r 4 -p 40 -w 4 \
          --minimum-trimmed-read-length 35 \
          --mask-short-adapter-reads 22 \
          --adapter-stringency 0.9 \
    --ignore-missing-bcls \
    --ignore-missing-filter \
    --ignore-missing-positions \
    --no-lane-splitting

and then I used cutadapt to remove the adapter

cutadapt -a CTGTCTCTTATACACATCT -A CTGTCTCTTATACACATCT -o SPG_rep3_trimmed_R1.fastq -p SPG_rep3_trimmed_R2.fastq  SPG_rep3_S7_R1_001.fastq.gz  SPG_rep3_S7_R2_001.fastq.gz

Is it possible this command affect the reads and cause the reduced properly mapping?

bowtie2 singletons
• 3,376 views • link
written by daffodil • daffodil

Did you check the reads quality before alignment? This looks like one of your pairs is failed or has a problem in read labels

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written by JC JC

1 answer

Uncertainty regarding the species of your data may be primarily due to the poor quality of the reference genome used.

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written by Huiyang • Huiyang

yes I have checked by the fastqc .enter link description here

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written by daffodil • daffodil

How does this address Huiyang's concern about the reference genome?

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written by Ram Ram

You can provide the download link for your reference genome, and I will help you check the quality of the reference genome.

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written by Huiyang • Huiyang

Sorry, you mean that I provide the reference genome (mm10) that I have used for alignment. It on the server and I used it directly from server.

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written by daffodil • daffodil

The mm10 genome is free of any issues. Your paired-end data is not genuinely paired, possibly due to errors during the bcl2fastq (bcl2fastq) or during other quality control procedures (cutadapt). You can focus on the labels of your paired reads to check if the read names are identical.

• 0 views • link
written by Huiyang • Huiyang

As far as adapter and quality control are concerned, I try to remove them using Fastp. As i mentioned I used this comands for bcl2fastq

bcl2fastq -R /proj/naiss2023-22-1174/Masomeh/240131_NB551428_0057_AHT2V3BGXV \
    --input-dir /proj/naiss2023-22-1174/Masomeh/240131_NB551428_0057_AHT2V3BGXV/Data/Intensities/BaseCalls/ \
    --output-dir /proj/naiss2023-22-1174/Masomeh \
    --interop-dir /proj/naiss2023-22-1174/Masomeh/240131_NB551428_0057_AHT2V3BGXV/Data/Intensities/InterOp/ \
    --stats-dir /proj/naiss2023-22-1174/Masomeh/240131_NB551428_0057_AHT2V3BGXV/Data/Intensities/BaseCalls/Stats/ \
    --reports-dir /crex/proj/naiss2023-22-1174/Masomeh/BaseCalls/Reports/ \
    --sample-sheet 'ATACsamplesheet.csv' \
-r 4 -p 40 -w 4 \
          --minimum-trimmed-read-length 35 \
          --mask-short-adapter-reads 22 \
          --adapter-stringency 0.9 \
    --ignore-missing-bcls \
    --ignore-missing-filter \
    --ignore-missing-positions \
    --no-lane-splitting

should I remove this part ?"

--minimum-trimmed-read-length 35 \
          --mask-short-adapter-reads 22 \
          --adapter-stringency 0.9 \
    --ignore-missing-bcls \
    --ignore-missing-filter \
    --ignore-missing-positions \"
• 0 views • link
updated by GenoMax GenoMax • written by daffodil • daffodil

Finally, I've figured it out. All alignment samples increased to 99% when I used fastp instead of cutadapt.

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written by daffodil • daffodil

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