Two mate pair libraries have 0 reads mapped during scaffolding

[Ray Cui]

Hello,

        I am currently doing de novo assembly with some old (>5years) illumina PE and MP data. An earlier version of the genome is already available, so we know the insert sizes and characteristics of each read library quite well. I determined the read orientation and insert size by mapping to the previous version of reference genome with BWA.

        The strange thing is that 2 libraries (with 50bp x 2 reads, phred33, MP) have 0 connections, while other libraries seem fine (100bp x 2, phred64/phred33, PE + MP). These 2 libraries are contaminated with normal PE reads (about 30% - 50%):

For insert size: 2134

 Total PE links                      0

 Normal PE links on same contig      0

 Incorrect oriented PE links         0

 PE links of too small insert size   0

 PE links of too large insert size   0

 Correct PE links                    0

 Accumulated connections             0

Use contigs longer than 2134 to estimate insert size: 

 PE links               0

Too few PE links.

0 new connections.

===========================

For insert size: 4886

 Total PE links                      0

 Normal PE links on same contig      0

 Incorrect oriented PE links         0

 PE links of too small insert size   0

 PE links of too large insert size   0

 Correct PE links                    0

 Accumulated connections             0

Use contigs longer than 4886 to estimate insert size: 

 PE links               0

Too few PE links.

0 new connections.

        These are the definitions I have in the config file for these two libraries:

[LIB]

avg_ins=2134.14

reverse_seq=1

asm_flags=2

rd_len_cutoff=1000

rank=4

pair_num_cutoff=4

map_len=32

q1=BGI2K_MP_1.fq.gz.trimmed.paired.fq.gz

q2=BGI2K_MP_2.fq.gz.trimmed.paired.fq.gz

[LIB]

avg_ins=4886.89

reverse_seq=1

asm_flags=2

rd_len_cutoff=1000

rank=5

pair_num_cutoff=4

map_len=32

q1=./BGI5K_MP_1.fq.gz.trimmed.paired.fq.gz

q2=./BGI5K_MP_2.fq.gz.trimmed.paired.fq.gz

            Could this be caused by the contaminating PE reads in the MP libraries or does it have something to do with the short read length?

Best Regards

Rongfeng Cui

[Ray Cui]

I think I figured it out, it's the -k parameter which is by default set to the same number as -K for the contigging stage. After changing it to be 35 it works.

Best

Ray