Dear All,

I have paired-end RNAseq data that were aligned to hg38 genome using hisat2 with strand-specific information --dta --rna-strandness RF option.

I have lncRNA transcript which is on a positive-strand and the start and end positions are 9:115046349-115047199. I have the bam files and wanted to extract reads on the positive strand for that specific region with +/- 5kb of that region.

I tried with samtools:

samtools view -F 16 -b sample.sorted.bam “9:115041349-115052199" > sample_specificregion.bam

I see that -F discard the reads mapping on the negative strand with the flag 16.

But I see the reads in sample_specificregion.bam were showing - strand

Is this flag -F 16 right way to get reads on the positive strand for that specific region?

D00535:34:CBMGRANXX:5:2109:12145:31393  99  9   115052134   60  92M =   115052157   120 AGAGGAAAATTCTGTCATTTTCAACAACACGGATGAACCCAGAAGACATTGTACAGAGTGAAATAATCGAGGCACAGAAAGACAAATACTGC    GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:92 YS:i:0  YT:Z:CP XS:A:-  NH:i:1
D00535:34:CBMGRANXX:5:2103:4351:30673   99  9   115052135   60  93M =   115052143   106 GAGGAAAATTCTGTCATTTTCAACAACACGGATGAACCCAGAAGACATTGTACAGAGTGGAATAATCGAGGCACAGAAAGACAAATACTGCGT   FBGGFGGBGGGGGGGGGGGEGGGGGGGGGGGCGGGGGGGGGGGGGGGGGEGGGGGGG<1FFGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGG   AS:i:-5 XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1  MD:Z:59A33YS:i:-5   YT:Z:CP XS:A:-  NH:i:1
D00535:34:CBMGRANXX:6:2110:18471:54222  99  9   115052135   60  93M =   115052316   278 GAGGAAAATTCTGTCATTTTCAACAACACGGATGAACCCAGAAGACATTGTACAGAGTGAAATAATCGAGGCACAGAAAGACAAATACTGCGT   GGGGGGGGGGGGGGGGGGGGGGGGGGGGBGGGGGGGGGGGFGGGGGGGGGGGGGGFGGGEGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGG   AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:93 YS:i:0  YT:Z:CP XS:A:-  NH:i:1
D00535:34:CBMGRANXX:6:2107:12093:34963  97  9   115052135   60  89M =   115052133   -94 GAGGAAAATTCTGTCATTTTCAACAACACGGATGAACCCAGAAGACATTGTACAGAGTGAAATAATCGAGGCACAGAAAGACAAATACT   GFGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG   AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:89 YS:i:0  YT:Z:DP XS:A:-  NH:i:1
D00535:34:CBMGRANXX:6:1301:21091:74975  99  9   115052135   60  93M =   115052143   106 GAGGAAAATTCTGTCATTTTCAACAACACGGATGAACCCAGAAGACATTGTACAGAGTGAAATAATCGAGGCACAGAAAGACAAATACTGCGT   FGGGGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGGFGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGEGG   AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:93 YS:i:-4 YT:Z:CP XS:A:-  NH:i:1
D00535:34:CBMGRANXX:6:1111:8020:42383   99  9   115052136   60  93M =   115052222   184 AGGAAAATTCTGTCATTTTCAACAACACGGATGAACCCAGAAGACATTGTACAGAGTGAAATAATCGAGGCACAGAAAGACAAATACTGCGTG   GGGGGGGGGGGGGGGGFGGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGEGGGGGGGGGGGGGGGGGGGGGCGGGG   AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:93 YS:i:0  YT:Z:CP XS:A:-  NH:i:1
D00535:34:CBMGRANXX:5:1109:7064:82839   99  9   115052152   60  92M =   115052152   98  TTTCAACAACACGGATGAACCCAGAAGACATTGTACAGAGTGAAATAATCGAGGCACAGAAAGACAAATACTGCGTGATCTCATTTGTGTAA    GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:92 YS:i:0  YT:Z:CP XS:A:-  NH:i:1
D00535:34:CBMGRANXX:5:1307:7118:29073   65  9   115052181   60  91M2S   1   66292253    0   ATTGTACAGAGTGAAATAATCGAGGCACAGAAAGACAAATACTGCGTGATCTCATTTGTGTAATCTAAGAAAGCTGAACACATAGAAGCACTC   GGGGGGGGGGGGGGFGGGEGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG   AS:i:-2 XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:91 YS:i:0  YT:Z:DP XS:A:-  NH:i:1
D00535:34:CBMGRANXX:6:2315:7732:93870   99  9   115052182   60  92M =   115052270   186 TTGTACAGAGTGAAATAATCGAGGCACAGAAAGACAAATACTGCGTGATCTCATTTGTGTAATCTAAGAAAGCTGAACACATAGAAGCACAG    GGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGFGGGGGGFGGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGGGGGGGGGEGGGGGG    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:92 YS:i:0  YT:Z:CP XS:A:-  NH:i:1