high rate of reads unmapped: too short

[z626093820]

When I use STAR for alignment, many reads do not align. How can I resolve this? THANK YOU !

below is my parameter:

STAR --runThreadN 15 --runMode alignReads --quantMode TranscriptomeSAM GeneCounts --sjdbOverhang 149 --twopassMode Basic --outSAMtype BAM SortedByCoordinate --outBAMsortingThreadN 15 --limitSjdbInsertNsj 12115510 --genomeDir /storage/zuozd/genome/ipomoea_batatas_tai6/star-gffreadgtf149/ --readFilesIn /storage/zuozd/20230921bleeding_sap/00cleandata/${i}_1.clean.fq.gz /storage/zuozd/20230921bleeding_sap/00cleandata/${i}_1.clean.fq.gz --readFilesCommand zcat --outFileNamePrefix ${i}

[z626093820]

When I use hisat2 comparison software, the comparison rate is high, why is it

[alexdobin]

It could be an issue with pairing of reads in FASTQ files. You can test it by mapping Read1 and Read2 separately.

[z626093820]

It could be an issue with pairing of reads in FASTQ files. You can test it by mapping Read1 and Read2 separately.

When I map the Read1 and Read2 files separately, the map rate of the two files reaches more than 80%，how can i slove the problem about the pairing of reads in FASTQ files? THANK YOU

[alexdobin]

If you are trimming reads before mapping, I would try to map untrimmed (raw) reads.

[z626093820]

If you are trimming reads before mapping, I would try to map untrimmed (raw) reads.

When I analyze the new command parameters given by the sequencing company, the mapping rate has reached 70%. Excuse me, which of these parameters have played a key role？THANK YOU!

the low mapping rate command : STAR --runThreadN 15 --runMode alignReads --quantMode TranscriptomeSAM GeneCounts --sjdbOverhang 149 --twopassMode Basic --outSAMtype BAM SortedByCoordinate --outBAMsortingThreadN 15 --limitSjdbInsertNsj 12115510 --genomeDir /storage/zuozd/genome/ipomoea_batatas_tai6/star-gffreadgtf149/ --readFilesIn /storage/zuozd/20230921bleeding_sap/00cleandata/${i}_1.clean.fq.gz /storage/zuozd/20230921bleeding_sap/00cleandata/${i}_1.clean.fq.gz --readFilesCommand zcat --outFileNamePrefix ${i}

the high mapping rate command: STAR --runThreadN 15 --runMode alignReads --readFilesCommand zcat --quantMode GeneCounts --outSAMtype BAM SortedByCoordinate --outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outFilterMismatchNmax 2 --outReadsUnmapped Fastx --twopassMode Basic --chimSegmentMin 12 --chimJunctionOverhangMin 12 --chimSegmentReadGapMax 3 --alignSJstitchMismatchNmax 5 -1 5 5 --chimOutJunctionFormat 1 --genomeDir /storage/zuozd/genome/ipomoea_batatas_tai6/star-gffreadgtf149/ --readFilesIn /storage/zuozd/20230921bleeding_sap/00cleandata/L58-1_1.clean.fq.gz /storage/zuozd/20230921bleeding_sap/00cleandata/L58-1_2.clean.fq.gz --outFileNamePrefix L58-1