Hello,
I have Paired End Data that I am trying to align to a very short reference(222 bp); I expect that the paired-end data have a lot of overlap.
When I run BWA-MEM on any of my samples, I get the following output:
[M::mem_pestat] # candidate unique pairs for (FF, FR, RF, RR): (0, 6, 0, 0)
[M::mem_pestat] skip orientation FF as there are not enough pairs
[M::mem_pestat] skip orientation FR as there are not enough pairs
[M::mem_pestat] skip orientation RF as there are not enough pairs
[M::mem_pestat] skip orientation RR as there are not enough pairs
Why am I getting so few read pairs? Could this be due to poor sequencing?
This is the first read of R1 & R2 so they are in order and labeled correctly in the header:
@MN01173:264:000H57WYJ:1:11101:14464:1123 1:N:0:ANATGTAGTA+CACCCGTA
CAAGGGGCCGCCCACCAGCCNCTGTTCATNNTGCTGCAGGTCCAGGGCCAGGTGCTGNCTNCANGAGTGGGGAACCAAACGCTGAAAGGAGCCAGAGTGATGTCATGACNGGAGAACNCCNGGACNTGGATTTAAGCAGCAGATCGGAAG
+
AFFFFFFFFFFFFAFFFFFA#AFFFFFFF##FFFFFFFFFFFAAFFFFFFFFFFFFF#FF#FF#FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFAFFFAFFFAAF#FFFFFFF#F=#AFFF#FFFFFFAFFFFFFFFFFFFFAFF/
r2
@MN01173:264:000H57WYJ:1:11101:14464:1123 2:N:0:ANATGTAGTA+CACCCGTA
GCTGCTTAAATCCAAGTCCTGGGGTTCTCCCGTCATGACATNACTCTGGCTCCTTTCAGCGTTNGGTTCCCCACTCCTGGAGTCAGCACNTGGCCCTGGANCTGCAGCACCATGAACAGTGGCTNGTGGGCGGCCCCTTGAGATCGGAAG
+
AFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF#FFFFFFFFFFFFFFFFFFFFA#FFFFFFFFFFFFFFFFFFFFFFAFF#FFFFFFFFFF#FFFFFFFFFF=FFFFFFFFFFFF#FFFFFFFFFFFFF=FFFF/FFFFF/
I also notice that some of my reads have long Adenine/Guanine Repeats in the reads and I ave never seen this before:
R1
GGCGGCCCCTTGTTTGATGTNCAGAGGGAGNTGAGATCGGAAGAGCACACGTCTGAANTCNAGNCACAGCTCACGTATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAANGGGGGGGNGGNGGGGNGGGGGGGGGGGGGGGGGGGGGGGG
R2
CACCTCCCTCTGCACATCAAACAAGGGGCCGCCAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTTGGATAAAGGTGTAGATCTCGGTNGTCGCCGTATCATTAAAAAAAAAAGGGGGGGGGGNGGGGGGGGGGGGGGGGGGGGGGGGG
This is my bwa-mem command:
bwa mem balv3.fa S21_R1_001.fastq.gz S21_R2_001.fastq.gz > S21_1314.sam
& this is the reference sequence:
GTACAAGACCCAACAACAATACAAGAAAAAGTATAAATATAGGACCAGGCAGAGCATTTTATACAACAGGAGAAATAATAGGAGATATAAGACAAGCACATTGTAACCTTAGTAGAGCAAAATGGAATGACACTTTAAATAAGATAGTTATAAAATTAAGAGAACAATTTGGGAATAAAACAATAGTCTTTAAGCACTCCTCAGGAGGGGACCCAGAAATTG
Thank You, Sara
