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Rangika 09-19-2016 08:37 PM

Selecting the best alignment BAM file

I have a PE dataset 300bp inserts by illumina MiSeq. I aligned the raw data using BWA-mem. Mapping statistics generated using Samtools flagstat are below.

5541008 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
76008 + 0 supplementary
0 + 0 duplicates
5413610 + 0 mapped (97.70% : N/A)
5465000 + 0 paired in sequencing
2732500 + 0 read1
2732500 + 0 read2
5266140 + 0 properly paired (96.36% : N/A)
5319406 + 0 with itself and mate mapped
18196 + 0 singletons (0.33% : N/A)
32368 + 0 with mate mapped to a different chr
8821 + 0 with mate mapped to a different chr (mapQ>=5)

I also used Trimmomatic on the same dataset, ILLUMINACLIP to remove any adapter sequences, trimmed reads sliding window 4:10, leading & trailing bases <3, length <39bp. Aligned this set using BWA-mem and got the results as below.

5529752 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
65642 + 0 supplementary
0 + 0 duplicates
5396698 + 0 mapped (97.59% : N/A)
5464110 + 0 paired in sequencing
2732055 + 0 read1
2732055 + 0 read2
5263982 + 0 properly paired (96.34% : N/A)
5308488 + 0 with itself and mate mapped
22568 + 0 singletons (0.41% : N/A)
23856 + 0 with mate mapped to a different chr
4865 + 0 with mate mapped to a different chr (mapQ>=5)

1) Can I use this information to select a best alignment based on mapped %. Raw data gave 97.7% mapping which is higher than trimmed data. So can I select BAM I got from raw data as the best?

2) I used "samtools view -c -f 3 data.bam" to find the properly paired reads. But the value I got is different to the value for that parameter by flagstat for both datasets. I checked some other parameters like itself & mate mapped they too gave different results. What could be the reason.

Appreciate your answers.
Thanks in advance.


Michael.Ante 09-20-2016 12:19 AM

Hi Rangika,

2nd first:
You need to be aware of the fact that samtools flagstat produces statistics on alignments. Meaning, a read can align multiple time and will occur multiple times in the flagstat output. You may check your alignment file with e.g. from the RSeQC tools.
Furthermore, I'd check the read files with FastQC before and after trimming.

1) I'd check a set of different data sets to choose which way to go. Also, I would not rely on the %mapped from samtools flagstat.


Rangika 09-20-2016 01:14 AM

Thank you Michael. My dataset is DNA-seq. Can I use RSeQC tools to check alignment for DNA data as well. Do you suggest RSeQC statistics would lead in to better BAM selection?

Appreciate if you would clarify this a bit more since I'm new to this.


Michael.Ante 09-20-2016 02:45 AM

The was a suggestion since it also works for DNA-seq alignments (you'll hopefully don't see spliced reads).
You can also have a look at the QC-metrics from Picard tools, or have a look at GATK.
Or you can extract the aligned reads (samtools view) and count e.g. how often each read is aligned. Without trimming you might have a high %mapping rate given by samtools flagstat, but you don't know how many reads were aligned with a high confidence to a single or few positions.

Most of the library preps have also a small section of how to deal with the analysis. Additionally, there are a plethora of publications describing their approach to DNA-Seq analysis.


Rangika 09-20-2016 08:29 PM

Thank you Michael for your answer.


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