Problem mapping with STAR

Question

[alvarocentron91]

Hello, I'm mapping with three different programs: TopHat, HISAT2, and STAR. I'm using default values for all but STAR (I have short reads after trimming):

#TopHat
tophat -I 300 -i 20 $HOME/Doct2.0/Genomes/Ustilago/BowTie2_index/Ustilago ../ax3_1_paired.fastq ../ax3_2_paired.fastq

#HISAT2
hisat2 -p 18 -x /home/acenbro/Doct2.0/Genomes/Ustilago/HISAT2_index/Ustilago -1 ../ax3_1_paired.fastq -2 ../ax3_2_paired.fastq -S ax3_HISAT2.sam

#STAR
STAR --runThreadN 18 --genomeDir $HOME/Doct2.0/Genomes/Ustilago/STAR_index/ --readFilesIn $HOME/Doct2.0/Data/ax_3/ax3_1_paired.fastq $HOME/Doct2.0/Data/ax_3/ax3_1_paired.fastq --outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 --outFilterMatchNmin 40

When I do samtools flagstat in each .bam I get logic results for TopHat and HISAT however with STAR I got a huge difference in the number of reads 1 and reads 2 and a really little number of properly paired reads:

#TopHat
72862137 + 0 in total (QC-passed reads + QC-failed reads)
665978 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
72862137 + 0 mapped (100.00% : N/A)
72196159 + 0 paired in sequencing
36254310 + 0 read1
35941849 + 0 read2
66340500 + 0 properly paired (91.89% : N/A)
70583534 + 0 with itself and mate mapped
1612625 + 0 singletons (2.23% : N/A)
1236274 + 0 with mate mapped to a different chr
1211542 + 0 with mate mapped to a different chr (mapQ>=5)

#HISAT2
77017110 + 0 in total (QC-passed reads + QC-failed reads)
882162 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
76025120 + 0 mapped (98.71% : N/A)
76134948 + 0 paired in sequencing
38067474 + 0 read1
38067474 + 0 read2
64971276 + 0 properly paired (85.34% : N/A)
74832130 + 0 with itself and mate mapped
310828 + 0 singletons (0.41% : N/A)
1615256 + 0 with mate mapped to a different chr

#STAR
77920325 + 0 in total (QC-passed reads + QC-failed reads)
39755776 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
77920325 + 0 mapped (100.00% : N/A)
38164549 + 0 paired in sequencing
37728565 + 0 read1
435984 + 0 read2
834538 + 0 properly paired (2.19% : N/A)
834538 + 0 with itself and mate mapped
37330011 + 0 singletons (97.81% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

I read the manual and I think I'm not seeing something and I'm feeling a little bit frustrated so any help would be great guys,

thank you in advance.

[WouterDeCoster]

You really shouldn't be using TopHat anymore, at least use Tophat2. And even that one is replaced by HISAT.

Please stop using Tophat https://t.co/Es4ohxOEyx Cole and I developed the method in *2008*. It was greatly improved in TopHat2 then HISAT & HISAT2. There is no reason to use it anymore. I have been saying this for years yet it has more citations this year than last #methodsmatter

— Lior Pachter (@lpachter) December 2, 2017

Answer 1

[WouterDeCoster]

$HOME/Doct2.0/Data/ax_3/ax3_1_paired.fastq $HOME/Doct2.0/Data/ax_3/ax3_1_paired.fastq

You use the same fastq twice for STAR.

[alvarocentron91]

Ok, I was too focus on parameters and I did that dumb mistake, thank you! And about TopHat I know, but I was taught with it even if it is not used anymore and I used it as a control to know that I did ok with the other programs

[Kevin Blighe]

Not dumb - no te preocupes. We have all done it before. Best wishes / Hasta luego

[alvarocentron91]

Now I have a problem visualizing the data in IGV, I'm getting several reads with huge insert sizes. I'm trying to solve it setting in -4 the --scoreInsOpen --scoreInsBase parameters, I think in this way I will penalize long inserts but I'm not sure due to my lack of knowledge in this field and don't find much information in the manual or internet, so If you can give me a tip on how to control the insert size would be great!

[GenoMax]

Not a good practice to ask unrelated questions in an existing thread. You should create a new question for this.

That said what do you mean by a tip on how to control the insert size?

[alvarocentron91]

Ok, I'll open a new thread. And I meant how to set a threshold for the maximum insert size