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